Host inflammatory dynamics reveal placental immune modulation by Group B Streptococcus during pregnancy.

Group B Streptococcus (GBS) is a pathobiont that can ascend to the placenta and cause adverse pregnancy outcomes, in part through production of the toxin ß-hemolysin/cytolysin (ß-h/c). Innate immune cells have been implicated in the response to GBS infection, but the impact of ß-h/c on their response is poorly defined. We show that GBS modulates innate immune cell states by subversion of host inflammation through ß-h/c, allowing worse outcomes. We used an ascending mouse model of GBS infection to measure placental cell state changes over time following infection with a ß-h/c-deficient and isogenic wild type GBS strain. Transcriptomic analysis suggests that ß-h/c-producing GBS elicit a worse phenotype through suppression of host inflammatory signaling in placental macrophages and neutrophils, and comparison of human placental macrophages infected with the same strains recapitulates these results. Our findings have implications for identification of new targets in GBS disease to support host defense against pathogenic challenge.

Multisystem Inflammatory Syndrome in U.S. Children and Adolescents.

BACKGROUND: Understanding the epidemiology and clinical course of multisystem inflammatory syndrome in children (MIS-C) and its temporal association with coronavirus disease 2019 (Covid-19) is important, given the clinical and public health implications of the syndrome. METHODS: We conducted targeted surveillance for MIS-C from March 15 to May 20, 2020, in pediatric health centers across the United States. The case definition included six criteria: serious illness leading to hospitalization, an age of less than 21 years, fever that lasted for at least 24 hours, laboratory evidence of inflammation, multisystem organ involvement, and evidence of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on reverse-transcriptase polymerase chain reaction (RT-PCR), antibody testing, or exposure to persons with Covid-19 in the past month. Clinicians abstracted the data onto standardized forms. RESULTS: We report on 186 patients with MIS-C in 26 states. The median age was 8.3 years, 115 patients (62%) were male, 135 (73%) had previously been healthy, 131 (70%) were positive for SARS-CoV-2 by RT-PCR or antibody testing, and 164 (88%) were hospitalized after April 16, 2020. Organ-system involvement included the gastrointestinal system in 171 patients (92%), cardiovascular in 149 (80%), hematologic in 142 (76%), mucocutaneous in 137 (74%), and respiratory in 131 (70%). The median duration of hospitalization was 7 days (interquartile range, 4 to 10); 148 patients (80%) received intensive care, 37 (20%) received mechanical ventilation, 90 (48%) received vasoactive support, and 4 (2%) died. Coronary-artery aneurysms (z scores ≥2.5) were documented in 15 patients (8%), and Kawasaki's disease-like features were documented in 74 (40%). Most patients (171 [92%]) had elevations in at least four biomarkers indicating inflammation. The use of immunomodulating therapies was common: intravenous immune globulin was used in 144 (77%), glucocorticoids in 91 (49%), and interleukin-6 or 1RA inhibitors in 38 (20%). CONCLUSIONS: Multisystem inflammatory syndrome in children associated with SARS-CoV-2 led to serious and life-threatening illness in previously healthy children and adolescents. (Funded by the Centers for Disease Control and Prevention.).

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity.

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits.

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

Vaginal carriage of Haemophilus influenzae in a non-pregnant reproductive-age population.

BACKGROUND: Haemophilus influenzae (Hi) is an emerging cause of early onset neonatal sepsis, but mechanisms of transmission are not well understood. We aimed to determine the prevalence of vaginal carriage of Hi in reproductive age women and to examine behavioral and demographic characteristics associated with its carriage. METHODS: We performed a secondary analysis of stored vaginal lavage specimens from a prospective cohort study of nonpregnant reproductive-age women. After extraction of bacterial genomic DNA, samples were tested for the presence of the gene encoding Haemophilus protein d (hpd) by quantitative real-time polymerase chain reaction (PCR) using validated primers and probe. PCR for the V3-V4 region of the 16 S rRNA gene (positive control) assessed sample quality. Samples with cycle threshold (CT) value < 35 were defined as positive. Sanger sequencing confirmed the presence of hpd. Behavioral and demographic characteristics associated with vaginal carriage of Hi were examined. RESULTS: 415 samples were available. 315 (75.9%) had sufficient bacterial DNA and were included. 14 (4.4%) were positive for hpd. There were no demographic or behavioral differences between the women with Hi vaginal carriage and those without. There was no difference in history of bacterial vaginosis, vaginal microbiome community state type, or presence of Group B Streptococcus in women with and without vaginal carriage of Hi. CONCLUSION: Hi was present in vaginal lavage specimens of 4.4% of this cohort. Hi presence was unrelated to clinical or demographic characteristics, though the relatively small number of positive samples may have limited power to detect such differences.

Cholera Vaccine: Recommendations of the Advisory Committee on Immunization Practices, 2022.

THIS REPORT SUMMARIZES ALL RECOMMENDATIONS FROM CDC'S ADVISORY COMMITTEE ON IMMUNIZATION PRACTICES (ACIP) FOR THE USE OF LYOPHILIZED CVD 103-HGR VACCINE (CVD 103-HGR) (VAXCHORA, EMERGENT BIOSOLUTIONS, GAITHERSBURG, MD) IN THE UNITED STATES. THE LIVE ATTENUATED ORAL CHOLERA VACCINE IS DERIVED FROM: Vibrio cholerae O1 and is administered in a single dose. Cholera is a toxin-mediated bacterial gastrointestinal illness caused by toxigenic V. cholerae serogroup O1 or, uncommonly, O139. Up to 10% of infections manifest as severe cholera (i.e., cholera gravis), profuse watery diarrhea that can cause severe dehydration and death within hours. Fluid replacement therapy can reduce the fatality rate to <1%. Risk factors for cholera gravis include high dose exposure, blood group O, increased gastric pH (e.g., from antacid therapy), and partial gastrectomy. Cholera is rare in the United States, but cases occur among travelers to countries where cholera is endemic or epidemic and associated with unsafe water and inadequate sanitation. Travelers might be at increased risk for poor outcomes from cholera if they cannot readily access medical services or if they have a medical condition that would be worsened by dehydration, such as cardiovascular or kidney disease. This report describes previously published ACIP recommendations about use of CVD 103-HgR for adults aged 18-64 years and introduces a new recommendation for use in children and adolescents aged 2-17 years. ACIP recommends CVD 103-HgR, the only cholera vaccine licensed for use in the United States, for prevention of cholera among travelers aged 2-64 years to an area with active cholera transmission. Health care providers can use these guidelines to develop the pretravel consultation for persons traveling to areas with active cholera transmission.

Group B Streptococcus Capsular Serotype Alters Vaginal Colonization Fitness.

BACKGROUND: Group B Streptococcus (GBS) remains a leading cause of infant morbidity and mortality. A candidate vaccine targets 6 GBS serotypes, offering a potential alternative to intrapartum antibiotic prophylaxis to reduce disease burden. However, our understanding of the contributions of specific capsule types to GBS colonization and disease remains limited. METHODS: Using allelic exchange, we generated isogenic GBS strains differing only in the serotype-determining region in 2 genetic backgrounds, including the hypervirulent clonal complex (CC) 17. Using a murine model of vaginal cocolonization, we evaluated the roles of the presence of capsule and of expression of specific capsular types in GBS vaginal colonization fitness independent of other genetic factors. RESULTS: Encapsulated wild-type strains COH1 (CC17, serotype III) and A909 (non-CC17, serotype Ia) outcompeted isogenic acapsular mutants in murine vaginal cocolonization. COH1 wild type outcompeted A909. Notably, expression of type Ia capsule conferred an advantage over type III capsule in both genetic backgrounds. CONCLUSIONS: Specific capsule types may provide an advantage in GBS vaginal colonization in vivo. However, success of certain GBS lineages, including CC17, likely involves both capsule and noncapsule genetic elements. Capsule switching in GBS, a potential outcome of conjugate vaccine programs, may alter colonization fitness or pathogenesis.

Do Not Forget About the Ticks: An Unusual Cause of Fever, GI Distress, and Cytopenias in a Child With ALL.

We report the case of a 5-year-old male with B-cell acute lymphoblastic leukemia in remission, receiving maintenance chemotherapy, who presented with fever, emesis, diarrhea, headache, and lethargy. He developed rapidly progressive cytopenias and was found to have acute human granulocytic anaplasmosis as well as evidence of past infection with Babesia microti. The case highlights the need to maintain a broad differential for infection in children undergoing chemotherapy or other immunosuppressive therapies with possible or known tick exposure.

Multisystem inflammatory syndrome in children.

PURPOSE OF REVIEW: Here we summarize current knowledge about multisystem inflammatory syndrome in children (MIS-C), a presumed postinfectious inflammatory condition that has emerged as an important COVID-19-associated complication, to help clinicians identify and manage cases. RECENT FINDINGS: Clinical presentation of MIS-C is dominated by significant inflammation. Fever, gastrointestinal symptoms, cardiac dysfunction, and hypotension are common features. Kawasaki disease-like findings are common, but epidemiologic data and recent mechanistic studies suggest that distinct inflammatory pathways mediate Kawasaki disease and MIS-C. A broad diagnostic approach is recommended, given overlapping presentations between MIS-C and many other disease processes. Current management of MIS-C is highly variable, depending on illness severity, and can range from supportive care to aggressive immune modulation. A multidisciplinary approach with early involvement of multiple pediatric subspecialists is recommended for complicated cases. SUMMARY: Several studies have described the clinical manifestations of MIS-C, but definitive diagnosis remains challenging. Robust information about long-term outcomes awaits further study, as do immunologic data to refine diagnostic and therapeutic strategies.

The Impact of Circulating Antibody on Group B Streptococcus Intestinal Colonization and Invasive Disease.

Gastrointestinal (GI) colonization with group B Streptococcus (GBS) is an important precursor to late-onset (LO) disease in infants. The host-pathogen interactions that mediate progression to invasive disease remain unknown due, in part, to a paucity of robust model systems. Passively acquired maternal GBS-specific antibodies protect newborns from early-onset disease, yet their impact on GI colonization and LO disease is unexplored. Using murine models of both perinatal and postnatal GBS acquisition, we assessed the kinetics of GBS GI colonization, progression to invasive disease, and the role of GBS-specific IgG production in exposed offspring and juvenile mice at age 12 and 14 days, respectively. We defined LO disease as >7 days of life in the perinatal model. We studied the impact of maternal immunization using a whole-cell GBS vaccine on the duration of intestinal colonization and progression to invasive disease after postnatal GBS exposure in offspring. Animals exhibit sustained GI colonization following both perinatal and postnatal exposure to GBS, with 21% and 27%, respectively, developing invasive disease. Intestinal colonization with GBS induces an endogenous IgG response within 20 days of exposure. Maternal vaccination with whole-cell GBS induces production of GBS-specific IgG in dams that is vertically transmitted to their offspring but does not decrease the duration of GBS intestinal colonization or reduce LO mortality following postnatal GBS exposure. Both perinatal and postnatal murine models of GBS acquisition closely recapitulate the human disease state, in which GBS colonizes the intestine and causes LO disease. We demonstrate both endogenous production of anti-GBS IgG in juvenile mice and vertical transfer of antibodies to offspring following maternal vaccination. These models serve as a platform to study critical host-pathogen interactions that mediate LO GBS disease.

Importance of Pediatric Inclusion in COVID-19 Therapeutic Trials.

Pediatric patients are excluded from most coronavirus disease 2019 (COVID-19) therapeutic trials. We outline a rationale for the inclusion of children in COVID-19 therapeutic trials, which enabled us to include children of all ages in a therapeutic COVID-19 trial at our institution.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Febrile Infants Without Respiratory Distress.

We report 2 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) in infants presenting with fever in the absence of respiratory distress who required hospitalization for evaluation of possible invasive bacterial infections. The diagnoses resulted from routine isolation and real-time reverse-transcription polymerase chain reaction-based testing for SARS-CoV-2 for febrile infants in an outbreak setting.

Multisystem Inflammatory Syndrome in Children Associated with Status Epilepticus.

A 12-year-old boy presented to the emergency department with findings concerning for multisystem inflammatory syndrome in children. After clinical stabilization following treatment with antibiotics, remdesivir, and anakinra, the patient was noted to have episodes of altered mentation. Video electroencephalogram revealed status epilepticus, which was subsequently controlled with antiepileptic medications.

Impact of Maternal Severe Acute Respiratory Syndrome Coronavirus 2 Detection on Breastfeeding Due to Infant Separation at Birth.

OBJECTIVE: To assess the impact of separation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR)-positive mother-newborn dyads on breastfeeding outcomes. STUDY DESIGN: This observational longitudinal cohort study of mothers with SARS-CoV-2 PCR-and their infants at 3 NYU Langone Health hospitals was conducted between March 25, 2020, and May 30, 2020. Mothers were surveyed by telephone regarding predelivery feeding plans, in-hospital feeding, and home feeding of their neonates. Any change prompted an additional question to determine whether this change was due to coronavirus disease-2019 (COVID-19). RESULTS: Of the 160 mother-newborn dyads, 103 mothers were reached by telephone, and 85 consented to participate. There was no significant difference in the predelivery feeding plan between the separated and unseparated dyads (P = .268). Higher rates of breastfeeding were observed in the unseparated dyads compared with the separated dyads both in the hospital (P < .001) and at home (P = .012). Only 2 mothers in each group reported expressed breast milk as the hospital feeding source (5.6% of unseparated vs 4.1% of separated). COVID-19 was more commonly cited as the reason for change in the separated group (49.0% vs 16.7%; P < .001). When the dyads were further stratified by symptom status into 4 groups-asymptomatic separated, asymptomatic unseparated, symptomatic separated, and symptomatic unseparated-the results remained unchanged. CONCLUSIONS: In the setting of COVID-19, separation of mother-newborn dyads impacts breastfeeding outcomes, with lower rates of breastfeeding both during hospitalization and at home following discharge compared with unseparated mothers and infants. No evidence of vertical transmission was observed; 1 case of postnatal transmission occurred from an unmasked symptomatic mother who held her infant at birth.

Group B streptococcal transmission rates as determined by PCR.

Background Group B Streptococcus (GBS) is a common cause of neonatal sepsis. GBS colonization of the newborn gastrointestinal tract (GIT) may be a critical precursor for late-onset infection. Assessment of the rate of neonatal GBS intestinal colonization has generally relied upon culture-based methods. We used polymerase chain reaction (PCR) and culture to determine the rate of GBS transmission to neonates. We hypothesized that PCR may enhance the detection of neonatal GBS colonization of the GIT, and that the rate will be higher when evaluated with PCR as compared to culture. Methods This was a cross-sectional study, in which mothers who were positive for GBS on routine screening and their healthy infants were eligible for recruitment. Newborn stool was collected after 24 h of life and before hospital discharge, and stored at -80°C for culture and PCR targeting the GBS-specific surface immunogenic protein (sip) gene. Results A total of 94 mother-infant pairs were enrolled; of these pairs, stool was collected from 83 infants. Based on PCR, the overall GBS transmission rate was 3.6% (3/83). The transmission rate was 2.4% (1/41) among vaginal deliveries and 4.8% (2/42) among cesarean deliveries. The results of culture-based transmission detection were identical. Conclusion These results indicate that the rate of GBS transmission is low and that detection may not be enhanced by PCR methods.

Storage Primes Erythrocytes for Necroptosis and Clearance.

BACKGROUND/AIMS: Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS: Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS: Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION: Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.

A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

High prevalence of Group B Streptococcus colonization among pregnant women in Amman, Jordan.

BACKGROUND: Little is known of the burden of Group B Streptococcus (GBS) colonization among pregnant women in Jordan. We conducted a pilot study to determine the prevalence of GBS among pregnant women in Amman, Jordan, where GBS testing is not routine. We also explored GBS serotypes and the performance of a rapid GBS antigen diagnostic test. METHODS: We collected vaginal-rectal swabs from women who presented for labor and delivery at Al-Bashir Hospital. Three methods were used to identify GBS: Strep B Rapid Test (Creative Diagnostics), blood agar media (Remel) with confirmed with BBL Streptocard acid latex test (Becton Dickinson), and CHROMagar StrepB (Remel). Results were read by a senior microbiologist. We defined our gold standard for GBS-positive as a positive blood agar culture confirmed by latex agglutination and positive CHROMagar. PCR testing determined serotype information. Demographic and clinical data were also collected. RESULTS: In April and May 2015, 200 women were enrolled with a median age of 27 years (IQR: 23-32); 89.0% were Jordanian nationals and 71.9% completed secondary school. Median gestational age was 38 weeks (IQR: 37-40); most women reported prenatal care (median 9 visits; IQR: 8-12). Median parity was 2 births (IQR: 1-3). Pre-pregnancy median BMI was 24.1 (IQR: 21.5-28.0) and 14.5% reported an underlying medical condition. Obstetric complications included gestational hypertension (9.5%), gestational diabetes (6.0%), and UTI (53.5%), of which 84.5% reported treatment. Overall, 39 (19.5%) of women were GBS-positive on blood agar media and CHROMagar, while 67 (33.5%) were positive by rapid test (36% sensitivity, 67% specificity). Serotype information was available for 25 (64%) isolates: III (48%), Ia (24%), II (20%), and V (8%). No demographic or clinical differences were noted between GBS+ and GBS-negative women. CONCLUSIONS: Nearly one in five women presenting for labor in Jordan was colonized with GBS, with serotype group III as the most common. The rapid GBS antigen diagnostic had low sensitivity and specificity. These results support expanded research in the region, including defining GBS resistance patterns, serotyping information, and risk factors. It also emphasizes the need for routine GBS testing and improved rapid GBS diagnostics for developing world settings.