Maternal HIV status skews transcriptomic response in infant cord blood monocytes exposed to Bacillus Calmette--Guerín.

OBJECTIVES: HIV-exposed uninfected (HEU) infants exhibit altered vaccine responses and an increased mortality compared with HIV-unexposed infants. Here, vaccine responses in HEU and HIV-unexposed cord blood monocytes (CBMs) were assessed following Bacillus Calmette--Guerín (BCG) treatment. DESIGN: Innate responses to in-vitro BCG treatment were assessed through transcriptional profiling using CBMs obtained from a Nigerian cohort of HIV-infected and uninfected women. METHODS: HIV-unexposed (n = 9) and HEU (n = 10) infant CBMs were treated with BCG and transcriptionally profiled with the Nanostring nCounter platform. Differential expression and pathway enrichment analyses were performed, and transcripts were identified with enhanced or dampened BCG responses. RESULTS: Following BCG stimulation, several pathways associated with inflammatory gene expression were upregulated irrespective of HIV exposure status. Both HIV-unexposed and HEU monocytes increased expression of several cytokines characteristic of innate BCG responses, including IL1ß, TNFα, and IL-6. Using differential expression analysis, we identified genes significantly upregulated in HEU compared with HIV-unexposed monocytes including monocyte chemokine CCL7 and anti-inflammatory cytokine TNFAIP6. In contrast, genes significantly upregulated in HIV-unexposed compared with HEU monocytes include chemokine CCL3 and cytokine IL23A, both of which influence anti-mycobacterial T-cell responses. Finally, two genes, which regulate prostaglandin production, CSF2 and PTGS2, were also more significantly upregulated in the HIV-unexposed cord blood indicating that inflammatory mediators are suppressed in the HEU infants. CONCLUSION: HEU monocytes exhibit altered induction of several key innate immune responses, providing mechanistic insights into dysregulated innate response pathways that can be therapeutically targeted to improve vaccine responses in HEU infants.

The Association Between Gestational Diabetes and Microbiota in Placenta and Cord Blood.

Objective: Early life is a critical period for gut microbial development. It is still controversial whether there is placental microbiota during a healthy pregnancy. Gestational diabetes mellitus (GDM) is associated with increased risk of metabolic syndrome in the offspring, and the mechanisms are unclear. We sought to explore whether microbiota in placenta and cord blood may be altered in GDM. Methods: Placenta and cord blood samples were collected from eight GDM and seven euglycemic (control) term pregnancies in cesarean deliveries without evidence of clinical infections. The Illumina MiSeq Sequencing System was used to detect the microbiota based on the V3-V4 hypervariable regions of the 16S ribosomal RNA gene. Results: The microbiota were detectable in all placental samples. Comparing GDM vs. controls, there were more operational taxonomic units (OTUs) (mean ± SE = 373.63 ± 14.61 vs. 332.43 ± 9.92, P = 0.024) and higher ACE index (395.15 ± 10.56 vs. 356.27 ± 8.47, P = 0.029) and Chao index (397.67 ± 10.24 vs. 361.32 ± 8.87, P = 0.04). The placental microbiota was mainly composed of four phyla: Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria at the phylum level and 10 dominant genera at the genus level in both GDM and controls. Despite the dominant similarity in microbiota composition, at the OTU level, the abundance of Ruminococcus, Coprococcus, Paraprevotella, and Lactobacillus were higher, whereas Veillonella was lower in the placentas of GDM vs. controls. The microbiota was detected in one of the 15 cord blood samples, and its components were similar as to the corresponding placental microbiota at both phylum and genus levels suggesting placental microbiota as the potential source. Conclusions: The most abundant phyla and genus of placental microbiota were similar in GDM and euglycemic pregnancies, but GDM was associated with higher diversity of placental microbiota. Further study is needed to confirm the existence of microbiota in cord blood in pregnancies without clinical infection.

Serological and molecular detection of Neospora caninum and Toxoplasma gondii in human umbilical cord blood and placental tissue samples.

Neosporosis primarily affects cattle and dogs and is not currently considered a zoonotic disease. Toxoplasmosis is a zoonosis with a worldwide distribution that is asymptomatic in most cases, but when acquired during pregnancy, it can have serious consequences. The seropositivity rates determined by the indirect fluorescent antibody test for Neospora caninum (N. caninum) and Toxoplasma gondii (T. gondii) were 24.3% (49 samples) and 26.8% (54 samples), respectively. PCR positivity for N. caninum was observed in two samples of cord blood (1%) using the Nc5 and ITS1 gene, positivity for T. gondii was observed in 16 samples using the primer for the B1 gene (5.5% positivity in cord blood and 2.5% positivity in placental tissue). None of the samples showed structures characteristic of tissue cysts or inflammatory infiltrate on histopathology. Significant associations were observed only between N. caninum seropositivity and the presence of domestic animals (p = 0.039) and presence of dogs (p = 0.038) and between T. gondii seropositivity and basic sanitation (p = 0.04). This study obtained important findings regarding the seroprevalence and molecular detection of N. caninum and T. gondii in pregnant women; however, more studies are necessary to establish a correlation between risk factors and infection.

Elimination of "kitome" and "splashome" contamination results in lack of detection of a unique placental microbiome.

BACKGROUND: A placental microbiome, which may be altered in gestational diabetes mellitus (GDM), has been described. However, publications raising doubts about the existence of a placental microbiome that is different than contaminants in DNA extraction kits and reagents ("kitomes") have emerged. The aims of this study were to confirm the existence of a placental microbiome distinct from contaminants and determine if it is altered in GDM mothers. RESULTS: We first enrolled normal weight, obese and GDM mothers (N = 17) at term elective cesarean section delivery in a pilot case control study. Bacterial DNA was extracted from placental parenchyma, maternal and cord blood, maternal vaginal-rectal swabs, and positive and negative controls with the standard Qiagen/MoBio Power Soil kit. Placentas had significantly higher copies of bacterial 16S rRNA genes than negative controls, but the placental microbiome was similar in all three groups and could not be distinguished from contaminants in blank controls. To determine the source and composition of the putative placental bacterial community identified in the pilot study, we expanded the study to 10 subjects per group (N = 30) and increased the number and variety of negative controls (N = 53). We modified our protocol to use an ultraclean DNA extraction kit (Qiagen QIAamp UCP with Pathogen Lysis Tube S), which reduced the "kitome" contamination, but we were still unable to distinguish a placental microbiome from contaminants in negative controls. We noted microbial DNA from the high biomass vaginal-rectal swabs and positive controls in placental and negative control samples and determined that this resulted from close proximity well-to-well cross contamination or "splashome". We eliminated this source of contamination by repeating the sequencing run with a minimum of four wells separating high biomass from low biomass samples. This reduced the reads of bacterial 16S rRNA genes in placental samples to insignificant numbers. CONCLUSIONS: We identified the problem of well-to-well contamination ("splashome") as an additional source of error in microbiome studies of low biomass samples and found a method of eliminating it. Once "kitome" and "splashome" contaminants were eliminated, we were unable to identify a unique placental microbiome.

Detection of microbial cell-free DNA in maternal and umbilical cord plasma in patients with chorioamnionitis using next generation sequencing.

BACKGROUND: Chorioamnionitis has been linked to spontaneous preterm labor and complications such as neonatal sepsis. We hypothesized that microbial cell-free (cf) DNA would be detectable in maternal plasma in patients with chorioamnionitis and could be the basis for a non-invasive method to detect fetal exposure to microorganisms. OBJECTIVE: The purpose of this study was to determine whether next generation sequencing could detect microbial cfDNA in maternal plasma in patients with chorioamnionitis. STUDY DESIGN: Maternal plasma (n = 94) and umbilical cord plasma (n = 120) were collected during delivery at gestational age 28-41 weeks. cfDNA was extracted and sequenced. Umbilical cord plasma samples with evidence of contamination were excluded. The prevalence of microorganisms previously implicated in choriomanionitis, neonatal sepsis and intra-amniotic infections, as described in the literature, were examined to determine if there was enrichment of these microorganisms in this cohort. Specific microbial cfDNA associated with chorioamnionitis was first detected in umbilical cord plasma and confirmed in the matched maternal plasma samples (n = 77 matched pairs) among 14 cases of histologically confirmed chorioamnionitis and one case of clinical chorioamnionitis; 63 paired samples were used as controls. A correlation of rank of a given microorganism across maternal plasma and matched umbilical cord plasma was used to assess whether signals found in umbilical cord plasma were also present in maternal plasma. RESULTS: Microbial DNA sequences associated with clinical and/or histological chorioamnionitis were enriched in maternal plasma in cases with suspected chorioamnionitis when compared to controls (12/14 microorganisms, p = 0.02). Analysis of the microbial cfDNA in umbilical cord plasma among the 1,251 microorganisms detectable with this assay identified Streptococcus mitis, Ureaplasma spp., and Mycoplasma spp. in cases of suspected chorioamnionitis. This assay also detected cfDNA from Lactobacillus spp. in controls. Comparison between maternal plasma and umbilical cord plasma confirmed these signatures were also present in maternal plasma. Unbiased analysis of microorganisms with significantly correlated signal between matched maternal plasma and umbilical cord plasma identified the above listed 3 microorganisms, all of which have previously been implicated in patients with chorioamnionitis (Mycoplasma hominis p = 0.0001; Ureaplasma parvum p = 0.002; Streptococcus mitis p = 0.007). These data show that the pathogen signal relevant for chorioamnionitis can be identified in both maternal and umbilical cord plasma. CONCLUSION: This is the first report showing the detection of relevant microbial cell-free cfDNA in maternal plasma and umbilical cord plasma in patients with clinical and/or histological chorioamnionitis. These results may lead to the development of a specific assay to detect perinatal infections for targeted therapy to reduce early neonatal sepsis complications.

Perinatal Gram-Positive Bacteria Exposure Elicits Distinct Cytokine Responses In Vitro.

During pregnancy, infections caused by the gram-positive bacteria Enterococcus faecalis (E. faecalis), Streptococcus agalacticae (S. agalacticae), and Staphylococcus aureus (S. aureus) are major reasons for preterm labor, neonatal prematurity, meningitis, or sepsis. Here, we propose cytokine responses to bacterial infections by the immature perinatal immune system as central players in the pathogenesis of preterm birth and neonatal sepsis. We aimed to close the gap in knowledge about such cytokine responses by stimulating freshly isolated umbilical blood mononuclear cells (UBMC) with lysates of E. faecalis, S. agalacticae, and S. aureus collected from pregnant women in preterm labor. Bacterial lysates and, principally, S. aureus and S. agalacticae distinctly triggered most of the eleven inflammatory, anti-inflammatory, TH1/TH2 cytokines, and chemokines quantified in UBMC culture media. Chemokines depicted the most robust induction. Among them, MIP-1ß was further enhanced in UBMC from female compered to male newborn infants. Due to its stability and high levels, we investigated the diagnostic value of IL-8. IL-8 was critically upregulated in cord blood of preterm neonates suffering from infections compared to gestational age-matched controls. Our results provide novel clues about perinatal immunity, underscoring a potential value of IL-8 for the timely detection of infections and suggesting that MIP-1ß constitutes an early determinant of sex-specific immunity, which may contribute, e.g., to male's vulnerability to preterm birth.

Análisis microbiológico del tracto genital materno y de la sangre del cordón umbilical en relación con el daño neonatal / Microbiological analysis of the maternal genital tract and umbilical cord blood and its association with neonatal damage

La etiología que conduce al daño neonatal es multifactorial, y los procesos infecciosos pueden estar implicados en él. El objetivo de este estudio fue identificar microorganismos del tracto genital materno asociados con el daño neonatal, a fin de prevenir futuras complicaciones perinatológicas. Se estudiaron 711 embarazadas que concurrieron entre enero de 2010 y julio 2013 al consultorio externo de Obstetricia del Hospital de Clínicas de la UBA para sus controles prenatales, y cuyos partos también tuvieron lugar en dicho nosocomio. En la sangre del cordón umbilical se investigó la presencia de Ureaplasma urealyticum y Mycoplasma hominis mediante el cultivo con sustratos metabólicos (Micofast-Biomerieux), y la de Trichomonas vaginalis por PCR, con primers específicos. El estudio microbiológico del contenido vaginal se efectuó en 288 de las embarazadas en la semana 35 a 37. Se empleó la metodología convencional, a la que se agregó el cultivo en tioglicolato modificado para T. vaginalis. Se investigó la presencia de estreptococos grupo B (EGB) en hisopado anorrectaly de introito vaginal, utilizando enriquecimiento en caldo selectivo y posterior siembra en medio cromogénico. Se utilizaron los test de χ² Yates y de Fisher para muestras independientes, considerándose significativo p < 0,05. La vaginosis bacteriana (VB) se relacionó significativamente con el daño neonatal (p = 0,02), al igual que la presencia de M. hominis (p = 0,03) y de T. vaginalis (p = 0,03) en la sangre del cordón umbilical. Las complicaciones predominantes fueron el parto pretérmino, la rotura prematura de membrana (RPM), el bajo peso y un valor de Apgar <7. No se asoció al daño neonatal la presencia de U. urealyticum (p = 0,35) en el cordón umbilical, ni la de Candidaspp. (p = 0,94) o EGB (p = 0,18) en el tracto genital de las madres. Dado que ciertas alteraciones en la microbiota del tracto genital materno se relacionaron con el dano neonatal, consideramos de fundamental importancia realizar el estudio microbiológico del contenido vaginal durante el embarazo, para prevenir posibles complicaciones maternas y perinatológicas.

The etiology leading to neonatal damage is multifactorial, being genital infections one of the causes. The objective of the study was to identify microorganisms of the maternal genital tract that are associated with neonatal damage, in order to prevent future perinatal complications. Seven hundred and eleven pregnant patients attended their prenatal control during the period January 2010-July 2013. Ureaplasma urealyticum and Mycoplasma hominis presence was investigated in umbilical cord blood by metabolic substrates (Micofast-Biomerieux) and that of T. vaginalis, by PCR using specific primers. The microbiological study of the vaginal contents of 288 pregnant patients at weeks 35 to 37 was performed by conventional methods, adding the modified thioglycolate culture for T. vaginalis. Group B streptococcus (GBS) was investigated in anorectal and vaginal introitus swabs, using selective broth enrichment and subsequent isolation in chromogenic medium. The χ² Yates test and Fisher's test were used for independent samples. A p value <0.05 was considered statistically significant. The pathogens significantly related to neonatal damage were M. hominis (p = 0.03), T. vaginalis (p = 0.03), and BV (p = 0.02). Main complications were preterm birth, premature rupture of membranes (PRM), low weight and Apgar score <7. U. urealyticum (p = 0.35), Candidaspp. (p = 0.94) and GBS (p = 0.18) were not related to neonatal damage. Since different microorganisms of the maternal genital tract were related to neonatal damage, it is very important to perform the microbiological study of vaginal contents during pregnancy to prevent possible maternal and perinatal complications.

Perinatal Ureaplasma Exposure Is Associated With Increased Risk of Late Onset Sepsis and Imbalanced Inflammation in Preterm Infants and May Add to Lung Injury.

Background: Controversy remains concerning the impact of Ureaplasma on preterm neonatal morbidity. Methods: Prospective single-center study in very low birth weight infants <30 weeks' gestation. Cord blood and initial nasopharyngeal swabs were screened for Ureaplasma parvum and U. urealyticum using culture technique and polymerase chain reaction. Neonatal outcomes were followed until death or discharge. Multi-analyte immunoassay provided cord blood levels of inflammatory markers. Using multivariate regression analyses, perinatal Ureaplasma exposure was evaluated as risk factor for the development of bronchopulmonary dysplasia (BPD), other neonatal morbidities until discharge and systemic inflammation at admission. Results: 40/103 (39%) infants were positive for Ureaplasma in one or both specimens, with U. parvum being the predominant species. While exposure to Ureaplasma alone was not associated with BPD, we found an increased risk of BPD in Ureaplasma-positive infants ventilated ≥5 days (OR 1.64; 95% CI 0.12-22.98; p = 0.009). Presence of Ureaplasma was associated with a 7-fold risk of late onset sepsis (LOS) (95% CI 1.80-27.39; p = 0.014). Moreover, Ureaplasma-positive infants had higher I/T ratios (b 0.39; 95% CI 0.08-0.71; p = 0.014), increased levels of interleukin (IL)-17 (b 0.16; 95% CI 0.02-0.30; p = 0.025) and matrix metalloproteinase 8 (b 0.77; 95% CI 0.10-1.44; p = 0.020), decreased levels of IL-10 (b -0.77; 95% CI -1.58 to -0.01; p = 0.043) and increased ratios of Tumor necrosis factor-α, IL-8, and IL-17 to anti-inflammatory IL-10 (p = 0.003, p = 0.012, p < 0.001). Conclusions: Positive Ureaplasma screening was not associated with BPD. However, exposure contributed to BPD in infants ventilated ≥5 days and conferred an increased risk of LOS and imbalanced inflammatory cytokine responses.

[Microbiological analysis of the maternal genital tract and umbilical cord blood and its association with neonatal damage]. / Análisis microbiológico del tracto genital materno y de la sangre del cordón umbilical en relación con el daño neonatal.

The etiology leading to neonatal damage is multifactorial, being genital infections one of the causes. The objective of the study was to identify microorganisms of the maternal genital tract that are associated with neonatal damage, in order to prevent future perinatal complications. Seven hundred and eleven pregnant patients attended their prenatal control during the period January 2010-July 2013. Ureaplasma urealyticum and Mycoplasma hominis presence was investigated in umbilical cord blood by metabolic substrates (Micofast-Biomerieux) and that of T.vaginalis, by PCR using specific primers. The microbiological study of the vaginal contents of 288 pregnant patients at weeks 35 to 37 was performed by conventional methods, adding the modified thioglycolate culture for T.vaginalis. GroupB streptococcus (GBS) was investigated in anorectal and vaginal introitus swabs, using selective broth enrichment and subsequent isolation in chromogenic medium. The χ2 Yates test and Fisher's test were used for independent samples. A p value <0.05 was considered statistically significant. The pathogens significantly related to neonatal damage were M.hominis (p=0.03), T.vaginalis (p=0.03), and BV (p=0.02). Main complications were preterm birth, premature rupture of membranes (PRM), low weight and Apgar score ≤7. U.urealyticum (p=0.35), Candidaspp. (p=0.94) and GBS (p=0.18) were not related to neonatal damage. Since different microorganisms of the maternal genital tract were related to neonatal damage, it is very important to perform the microbiological study of vaginal contents during pregnancy to prevent possible maternal and perinatal complications.

Can Fetal Umbilical Venous Blood Be a Reliable Source for Admission Complete Blood Count and Culture in NICU Patients?

BACKGROUND: Minimizing initial neonatal blood draws and their associated pain is important. The placenta has ample fetal blood that is otherwise discarded; obtaining admission laboratory evaluations from fetal umbilical venous blood (FUVB) may provide a suitable alternative. OBJECTIVE: We hypothesized that obtaining an aerobic bacterial blood culture (BCX) and a complete blood count with manual differential (CBC/diff) from FUVB is feasible and yields results comparable to those obtained directly from the neonate. STUDY DESIGN: BCX and CBC/diff were attempted on paired samples from FUVB (in the delivery room) and neonatal blood (shortly after NICU admission) of 110 patients. The paired t test, Pearson's correlation coefficient (R), and multivariable linear regression were used for data analysis. RESULTS: Positive BCXs were found in 9 of 108 FUVB samples compared to 1 of 91 neonatal samples. Three out of 9 FUVB cultures were true pathogens, including 2 Escherichia coli and 1 viridans group streptococcus, all with negative corresponding paired neonatal cultures. There was 1 positive neonatal BCX, E. coli, with a negative paired FUVB culture. Neonatal hemoglobin (Hb), platelets (PLT), and white blood cells (WBC) all significantly (p < 0.0001) correlated with the paired FUVB samples (R = 0.50, 0.49, and 0.84, respectively). Hb, PLT, and WBC values were clinically comparable but statistically higher in neonatal blood (the differences were 2.3 g/dL, 30,000 cells/µL, and 2,800 cells/µL, respectively; p < 0.007 for all comparisons). CONCLUSIONS: FUVB is suitable for obtaining CBC/diff. FUVB is an appropriate second source for BCX as it yields additional true pathogens. Our findings may support the presence of "culture-negative sepsis" in some neonates.