Meeting Report: WHO Workshop on modelling global mortality and aetiology estimates of enteric pathogens in children under five. Cape Town, 28-29th November 2018.

Investment in vaccine product development should be guided by up-to-date and transparent global burden of disease estimates, which are also fundamental to policy recommendation and vaccine introduction decisions. For low- and middle-income countries (LMICs), vaccine prioritization is primarily driven by the number of deaths caused by different pathogens. Enteric diseases are known to be a major cause of death in LMICs. The two main modelling groups providing mortality estimates for enteric diseases are the Institute for Health Metrics and Evaluation (IHME) at the University of Washington, Seattle and the Maternal Child Epidemiology Estimation (MCEE) group, led by Johns Hopkins Bloomberg School of Public Health. Whilst previous global diarrhoea mortality estimates for under five-year-olds from these two groups were closely aligned, more recent estimates for 2016 have diverged, particularly with respect to numbers of deaths attributable to different enteric pathogens. This has impacted prioritization and investment decisions for vaccines in the development pipeline. The mission of the Product Development for Vaccines Advisory Committee (PDVAC) at the World Health Organisation (WHO) is to accelerate product development of vaccines and technologies that are urgently needed and ensure they are appropriately targeted for use in LMICs. At their 2018 meeting, PDVAC recommended the formation of an independent working group of subject matter experts to explore the reasons for the difference between the IHME and MCEE estimates, and to assess the respective strengths and limitations of the estimation approaches adopted, including a review of the data on which the estimates are based. Here, we report on the proceedings and recommendations from a consultation with the working group of experts, the IHME and MCEE modelling groups, and other key stakeholders. We briefly review the methodological approaches of both groups and provide a series of proposals for investigating the drivers for the differences in enteric disease burden estimates.

[Increased sympathetic activity assessed by spectral analysis of heart rate variability in patients with CRPS I]. / CRPS I: Erhöhte Sympathikusaktivität in der Spektralanalyse der Herzfrequenzvariabilität.

PURPOSE: The complex regional pain syndrome type I (CRPS I) is a painful neuropathic disorder with an antecedent disproportionate trauma leading to spontaneous pain, hyperalgesia, impaired motor function, swelling, changes in sweating and vascular abnormalities without nerve injury. Whether this syndrome is the result of central or peripheral autonomic dysfunction is still a matter of debate. The purpose of this study was to determine the activity of the sympathetic nervous system in patients with CRPS I by power spectral analysis of heart rate variability. PATIENTS AND METHODS: This is a pilot study on 6 patients (mean age 50 years; 4 female, 2 male) diagnosed as suffering from CRPS I and 6 age-matched healthy controls. In the pain-free interval and after taking rest for 5 min, 512 subsequent heart beats were obtained with an ECG standard lead II in the supine and then sitting position. Using an autoregressive model, power spectral densities were calculated for the following frequency bands: <0.040 Hz (very low frequency; VLF), 0.040-0.150 Hz (low frequency; LF) and 0.150-0.4 Hz (high frequency; HF). The sympatho-vagal balance is expressed by the ratio of the low-frequency component (LF) to the high-frequency component (HF) of the power spectrum. RESULTS: Significant differences in the mean LF/HF ratios were found in the patients with CRPS I compared to the healthy controls in the supine position (LF/HF=4.01 vs. LF/HF=1.27; p=0.041). The application of stress by changing to the sitting position even increased that difference (6.72 vs. 1.93). CONCLUSIONS: Our results support the hypothesis that the pathogenesis of the early stage CRPS I might be related to an increased sympathetic activity. By assessing the autonomic influence on the heart rate variability in CRPS I patients we could also conclude that this disturbance occurs rather at a central level.

Cord blood banks collect units with different HLA alleles and haplotypes to volunteer donor banks: a comparative report from Swiss Blood stem cells.

Allogeneic haematopoietic SCT is a standard therapy for many patients with haematological diseases. A major aim of public umbilical cord blood (UCB) banking is to establish an inventory with a large HLA diversity. Few studies have compared HLA diversity between UCB banks and volunteer unrelated donor (VUD) registries and examined whether UCB banks indeed collect more units with rare alleles and haplotypes. This study compares HLA-A/B/DRB1 allele frequencies and inferred A/B/DRB1-haplotypes in 1602 UCB units and 3093 VUD from two centres in distinct recruitment areas in Switzerland. The results show that the frequencies of HLA-DRB1 alleles as well as of the HLA-A/B/DRB1 haplotypes differ between UCB and VUD. Ten DRB1 alleles occurred at a 2- to 12-fold higher relative frequency in UCB than in VUD and 27 rare alleles were identified in UCB. Out of these 27 alleles, 15 were absent in the entire VUD data set of the national registry. This difference in allele frequencies was found only by intermediate/high-resolution typing. Targeted recruitment of UCB units from non-Caucasian donors could further increase HLA allele and haplotype diversity of available donors. Intermediate or high-resolution DNA typing is essential to identify rare alleles or allele groups.

Quality management of nuchal translucency measurement in residents.

PURPOSE: The measurement of the nuchal translucency (NT) in the 1st trimester is a sensitive, reliable method to assess the risk of specific fetal chromosomal and other defects. Training, however, is an issue not only among experienced sonographers, but especially for ObGyn residents, since all NT measurements in a true screening setting should fulfil the quality standards. The aim of this study was therefore the evaluation of the learning curve of residents and determination of the number of measurements necessary to achieve acceptable results. MATERIALS AND METHODS: Between 30th June, 1997 and 8th August, 2003, we included 4450 subsequent pregnant women between 11+0 and 13+6 weeks of gestation referred for an NT scan and prenatal counselling (low and high risk patients) in the study. For analysis of the learning curve in residents, all NT scans performed either by the experienced sonographers only or by residents with less than 70 scans at the end of their training were excluded. As the main quality criterion, the percentage of cases above the median was used. To test for normal distribution of NT scans, the Kolmogorov-Smirnov test was used. RESULTS: Each of 19 residents fulfilling these criteria performed 131 NT scans (73-242) on average. 13 of 19 residents ultimately met the quality criteria, but the majority of residents achieved good quality only after 100 scans, whereas 6 of 19 never did. Only after at least 50 NT scans, 50% of measurements were above the median, whereas before these 50 scans, NT was usually underestimated. CONCLUSION: It became obvious that regular supervision and quality control is mandatory to provide exact NT measurements by residents. Based on our results, a minimum of 100 NT scans is recommended before diagnostic application, which is a higher requirement than implemented in widely accepted quality assurance programs.

Variations in policies for management of the third stage of labour and the immediate management of postpartum haemorrhage in Europe.

BACKGROUND: The EUropean Project on obstetric Haemorrhage Reduction: Attitudes, Trial, and Early warning System (EUPHRATES) is a set of five linked projects, the first component of which was a survey of policies for management of the third stage of labour and immediate management of postpartum haemorrhage following vaginal birth in Europe. OBJECTIVES: The objectives were to ascertain and compare policies for management of the third stage of labour and immediate management of postpartum haemorrhage in maternity units in Europe following vaginal birth. DESIGN: Survey of policies. SETTING: The project was a European collaboration, with participants in 14 European countries. SAMPLE: All maternity units in 12 countries and in selected regions of two countries in Europe. METHODS: A postal questionnaire was sent to all or a defined sample of maternity units in each participating country. MAIN OUTCOME MEASURES: Stated policies for management of the third stage of labour and the immediate management of postpartum haemorrhage. RESULTS: Policies of using uterotonics for the management of the third stage were widespread, but policies about agents, timing, clamping and cutting the umbilical cord and the use of controlled cord traction differed widely. For immediate management of postpartum haemorrhage, policies of massaging the uterus were widespread. Policies of catheterising the bladder, bimanual compression and in the choice of drugs administered were much more variable. CONCLUSIONS: Considerable variations were observed between and within countries in policies for management of the third stage of labour. Variations were observed, but to a lesser extent, in policies for the immediate management of postpartum haemorrhage after vaginal birth. In both cases, policies about the pharmacological agents to be used varied widely.

Both maternal and fetal cell-free DNA in plasma fluctuate.

Elevations in the concentration of cell-free fetal DNA in maternal plasma have recently been determined in various pregnancy-related disorders, including preeclampsia, preterm labor, and polyhydramnios. In addition, almost 2-fold increments in cell-free fetal DNA levels have been recorded in pregnancies with certain aneuploid fetuses, in particular trisomy 21. These findings have led to the speculation that quantitative assessment of circulatory fetal DNA may be useful in the noninvasive prenatal diagnosis of certain fetal genetic constellations or pregnancy-related disorders. A premise for any quantitative analysis is that the quantity of the analyte being assayed does not vary greatly over time. As this aspect has not been examined for circulatory DNA levels, we examined these in normal healthy individuals as well as in pregnant women. Our data indicate that severalfold alterations in circulatory DNA amounts do occur over short periods of time. Of particular note is that we observed almost 2-fold variations in free fetal DNA levels over a period of 3 days, which are in a similar range to the elevations noted in aneuploid pregnancies. Our results, therefore, imply that caution should be used when using small increments in circulatory fetal DNA concentrations for potential diagnostic applications.

Cell-free DNA in urine: a marker for kidney graft rejection, but not for prenatal diagnosis?

Intrigued by the rapid clearance of free fetal DNA from the maternal circulation, we have investigated whether this fetal genetic material could be cleared via the kidney. For this purpose, we examined for the presence of Y chromosome-specific DNA sequences in urine samples obtained from 8 women pregnant with male fetuses. No male-specific sequences could be detected, despite the use of a very sensitive nested PCR assay nor a highly reproducible real-time PCR assay. We did, however, detect maternal DNA sequences. To determine if this cell-free DNA was derived from the kidney or another source, we next examined urine from female kidney transplant patients who had received male kidneys. Y chromosome-specific sequences were indeed detectable by both nested and real-time PCR in these samples, thereby confirming a recent report describing urinary DNA microchimerism. Quantitative analysis of serially obtained samples furthermore suggests that transplant-derived sequences are elevated during periods of graft rejection. These results imply that the measurement of graft-derived urinary DNA may serve as a new marker for kidney graft tolerance.

[Prenatal diagnosis and management in VACTERL association]. / Pränatale Diagnose und Management bei VACTERL-Assoziation.

In terms of the VACTERL-Association we are dealing with a non-random association of malformations following a defect during mesodermal development of embryogenesis due to a variety of causes. We report on three cases with VACTERL-type malformations diagnosed by prenatal ultrasound presenting cardial defects, renal abnormalities, single umbilical arteries and esophageal stenosis. We present sonographical, clinical and autopsy findings and discuss the pathogenesis of VACTERL-Association as a defect of mesenchymal development in early embryogenesis.

Severely reduced presence of tissue macrophages in the basal plate of pre-eclamptic placentae.

Pre-eclampsia is a disorder of unknown aetiology peculiar to human pregnancy. A well-described pathological feature being shallow trophoblast invasion into the spiral arteries during placenta development. Epidemiological studies have revealed an increased risk in pregnancies of primipaternity, and an association with the maternal-fetal HLA-DR relationship, both suggesting the involvement of an immunological component. We were therefore interested in the distribution of HLA-DR expressing myeloid cells in the decidua of healthy and pre-eclamptic placentae. We have studied the monocytes in maternal and fetal peripheral blood as well as in the placenta and identified the cluster of differentiation (CD) 14(+)myeloid cells in the basal plate as mannose receptor (ManR) positive tissue macrophages. In a comparison between peripheral blood monocytes from healthy pregnant and pre-eclamptic women we found no significant difference in the subpopulation size of CD14(+)/CD16(+)monocytes. The number and location of macrophages in the placental villi was similar. However, while the basal plate of the normal decidua contained numerous CD14(+), HLA-DR(bright), ManR(+)tissue macrophages, this compartment was virtually void of these phagocytic cells in the pre-eclamptic placenta. This novel finding suggests that in pre-eclampsia not only the migration of endovascular cytotrophoblasts is disturbed, but that also maternal macrophage migration is affected.

Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma.

OBJECTIVE: To examine whether concentrations of free extracellular fetal circulatory DNA in maternal plasma are stable or fluctuate. METHODS: Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). RESULTS: In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. CONCLUSION: The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.

Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses.

Current non-invasive screening methods for the prenatal diagnosis of fetal aneuploidies are hampered by low sensitivities and high false positive rates. Attempts to redress this situation include the enrichment of fetal cells from maternal blood, or the use of fetal DNA in the plasma of pregnant women. By the use of real-time quantitative polymerase chain reaction (PCR) it has recently been shown that circulatory male fetal DNA in maternal plasma is elevated in pregnancies with trisomy 21 fetuses. In this independent study we confirm and extend upon these results by showing that the levels of fetal DNA are also elevated in pregnancies with other chromosomal aneuploidies (mean=185.8 genome equivalents/ml; range=62.2-471.7) when compared to pregnancies with normal male fetuses (mean=81.9 genome equivalents/ml; range=28.8-328.9), p=0.005. This elevation was greatest for fetuses with trisomy 21, whereas it was not significant for fetuses with trisomy 18, p=0.356. These data suggest that a quantitative analysis of such fetal DNA levels may serve as an additional marker for certain fetal chromosomal abnormalities, in particular for trisomy 21.

Current applications of single-cell PCR.

The advent of the polymerase chain reaction (PCR) has revolutionised the way in which molecular biologists view their task at hand, for it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell. It is especially in the field of prenatal diagnostics that this ability has been readily seized upon, as it has opened up the prospect of preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders. However, apart from diagnostic applications, single-cell PCR has proven to be of enormous use to basic scientists, addressing diverse immunological, neurological and developmental questions, where both the genome but also messenger RNA expression patterns were examined. Furthermore, recent advances, such as optimised whole genome amplification (WGA) procedures, single-cell complementary DNA arrays and perhaps even single-cell comparative genomic hybridisation will ensure that the genetic analysis of single cells will become common practice, thereby opening up new possibilities for diagnosis and research.

Multiplex and real-time quantitative PCR on fetal DNA in maternal plasma. A comparison with fetal cells isolated from maternal blood.

Fetal DNA has recently been detected in maternal plasma by PCR and has shown promise for the prenatal determination of fetal sex or rhesus D. In order to obtain the maximum amount of information from this fetal genetic material, we have devised a sensitive multiplex PCR method to permit simultaneous analysis for both the SRY locus and the rhesus D gene. Our studies show that this technique is very sensitive and specific. In the 22 cases from rhesus D negative women examined, we were able to determine both fetal genotypes correctly. In the parallel enrichment for fetal cells, fetal erythroblasts were only detected in 14 of the 19 cases. Our data also indicate that fetal DNA from rhesus D positive fetuses is present in maternal plasma even after prophylactic anti-D treatment. Furthermore, since fetal cells have been reported to be elevated in pregnancies with aneuploid fetuses, we have quantified the amount of fetal DNA present in the maternal plasma of 10 such affected pregnancies by real-time PCR. Our results indicate that fetal DNA is elevated under such circumstances when compared to gestationally matched normal pregnancies (mean of 7% in aneuploid samples versus 3.5% in normal pregnancies). These results indicate that the quantification of fetal DNA in maternal plasma may be an additional screening tool for pregnancies at risk of bearing an aneuploid fetus.

Choice of anticoagulant can influence the analysis using fluorescence in situ hybridization of fetal cells enriched from maternal blood.

A notable degree of research attention is being focused on the use of fetal cells enriched from the blood of pregnant women as a non-invasive means of prenatal diagnosis. By using magnetic activated cells sorting (MACS) and fluorescence in situ hybridization (FISH), we have examined the efficacy of enriched fetal cells in determining fetal sex. An unexpected finding of this investigation was that the sensitivity of this analysis was influenced by the anticoagulant used to treat the maternal blood samples. As such, samples treated with heparin showed significantly lower detection rates than samples chelated with EDTA.

Approximately half of the erythroblasts in maternal blood are of fetal origin.

The enrichment of fetal erythroblasts from the peripheral blood of pregnant women is currently actively pursued for the development of a non-invasive means of prenatal diagnosis. Since erythroblasts in maternal blood are not all of fetal origin, and currently no reliable method exists to distinguish between the maternal and fetal erythroblasts, their use for prenatal diagnosis is not without uncertainty. The purpose of this study was to determine the percentage of fetal erythroblasts in maternal blood at the single cell level and to what extent such cells can reproducibly be used for polymerase chain reaction (PCR)-based prenatal diagnostic analyses. Erythroblasts were enriched from the peripheral blood of rhesus negative pregnant women using magnetic cell sorting (MACS). Single erythroblasts identified morphologically were individually micromanipulated and analysed by a multiplex PCR reaction for the fetal SRY and rhesus D genes. As a control for the PCR reaction the beta-globin gene was used. The PCR results were validated by the results obtained by invasive procedures. In all instances where single erythroblasts were examined, the correct fetal genotype for the two fetal specific loci was detected. Furthermore, our results indicate that approximately 50% of the enriched erythroblasts are of fetal origin.

A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS.

The enrichment of fetal cells, in particular fetal erythroblasts from the blood of pregnant women offers a promising non-invasive alternative for prenatal diagnosis. The purpose of this study was to compare the retrieval of erythroblasts by different density gradients and different antibodies against erythroid surface antigens, in both a model test system and in blood samples of pregnant women. We enriched erythroblasts from artificial mixtures of cord and adult blood (1:50) and from 16 ml of peripheral blood from pregnant women at a mean gestational age of 14+2 weeks. The yield of erythroblasts was calculated and compared using Wilcoxon's matched-pairs signed-rank test. In the artificial mixture most erythroblasts were retrieved using the heaviest density gradient (specific density of 1119) followed by antibody-labelled magnetic cell sorting (MACS). With anti-GPA the yield of erythroblasts from the artificial mixture was highest (9362.5 erythroblasts) compared with anti-CD36 (5164.3), anti-i (3455.2), anti-CD71 (3055.8) and HAE9 (2364.2). The difference between anti-GPA and anti-CD71, HAE9 and anti-i was significant (p=0.0277). The enrichment of erythroblasts from peripheral blood of pregnant women showed similar results. The yield of erythroblasts using anti-GPA was the highest. These results enable us to simplify our enrichment protocols to a single density gradient of 1119 specific density followed by MACS, with anti-GPA.

Fetal cells in maternal blood. An update from Basel.

OBJECTIVE: The efficiency of two protocols for the enrichment of fetal cells from the blood of pregnant women was compared: a triple density gradient followed by twin magnetic separations (method A) versus a single density gradient and single magnetic separation (method B). STUDY DESIGN: Blood samples were obtained from women prior to undergoing an invasive procedure. The processed samples, 87 by method A and 332 by method B, were examined for the presence of male cells by fluorescence in situ hybridisation. RESULTS: The simpler protocol was found to be superior. The most critical component, however, is the ability of the reader to correctly evaluate the sample, where we observed large variations, with reader B attaining a sensitivity of 82.61% with a corresponding specificity of 86.96%. CONCLUSION: A simpler enrichment protocol can be used from smaller blood samples to attain detection efficiencies which are similar to or superior to current noninvasive methods.

[Validity of cardiotocography in the detection of umbilical cord complications]. / Zur Validität der Kardiotokographie bei Nabelschnurkomplikationen.

OBJECTIVE: The purpose of this study was to investigate the validity of cardiotocography for the detection of cord complications. MATERIAL AND METHODS: A low-risk population of 4196 cases was selected in which cord complications have been recognized in 34.3%. Cases with cord complications and controls were paired by parity, gestational age, maternal age and mode of delivery. 25 pairs were randomly selected. 50 tracings were presented twice to 4 obstetricians in a double-blind manner. As parameters for the determination of the validity of fetal monitoring the reliability, positive (ppv) and negative predictive value (npv), sensitivity and specificity were used. Inter- and intra-observer variability were also examined. RESULTS: Reliability 52%, ppv 52%, npv 52%, sensitivity 46%, specificity 58%. Interobserver variability: All 4 obstetricians agreed in 47 of 100 evaluations. The level of agreement was higher in the controls (63%) than in the cord complication group (56%). The intraobserver variability was 25%. CONCLUSIONS: Cardiotocography is not useful for the detection of cord complications. The range of possibilities has not been exploited yet, even for the evaluation of the fetal state.

[Prenatal diagnosis with fetal cells in maternal blood: report of experiences in Basal]. / Pränatale Diagnostik mittels fetaler Zellen im mütterlichen Blut: ein Erfahrungsbericht aus Basel.

Currently prenatal diagnosis relies on invasive procedures such as chorion villus sampling (CVS) or amniocentesis (AC). Many parents are reluctant to expose themselves and their child to the small, but significant risk posed by these procedures to mother and child. There is, hence, a great need for a risk-free non-invasive alternative. To achieve this goal most research has been focussed on enriching fetal cells from the blood of pregnant women. The erythroblast has emerged as the target cell of choice, since it is abundant in the early fetus, rare in normal adult blood, and since it has a very short half life, there is no risk of obtaining cells from previous pregnancies. Most enrichment protocols rely either on magnetic- or fluorescent activated cell sorting (MACS and FACS) using fetal specific antibodies. These enriched cells can be examined by FISH (fluorescence in-situ hybridisation) for the presence of the most common fetal chromosomal aneuploidies (13, 18, 21, X and Y) or by polymerase chain reaction (PCR) on singly manipulated cells for genetic disorders. The efficacy in detecting fetal aneuploidies is currently being evaluated in a phase II clinical trial under the auspices of the NIH-NICHD, the so-called NIFTY Trial, in which our group is a participant. By modifying our enrichment protocols we have recently been able to obtain detection sensitivities of almost 80%, thereby renewing our optimism that this methodology provides a solid basis for an effective non-invasive prenatal diagnostic test.

Prenatal diagnosis using fetal cells enriched from maternal blood.

The ability to use fetal cells enriched from the blood of pregnant women for prenatal diagnosis has been a long-sought goal for those pursuing a non-invasive alternative to current methods, such as amniocentesis or chorionic villus sampling. Several new developments, which rely either on fluorescence in situ hybridization or the combination of single-cell manipulation and subsequent polymerase chain reaction practices, which are common to the field of preimplantation genetics, have been tested. We discuss the significance of these developments and the obstacles that still have to be surmounted before this technology becomes available in a broad diagnostic use. The research in the field yielded important observations regarding the traffic of cells between the fetus and the mother, which may provide a new insight into the development of disorders such as preeclampsia and the associated HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome.