A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Hydrops fetalis and neonatal death from human parvovirus B19: an unusual complication.

A case of prenatally diagnosed human parvovirus B19 (HPVB19) infection is reported. The neonate died after intrauterine therapy and premature delivery. The fetus was diagnosed with oedema, cardiomegaly, poor myocardial contractility and a pericardial effusion at 24/40 weeks' gestation. Ultrasound using colour flow Doppler showed a midcerebral artery peak systolic velocity (MCA PSV) raised at 45 cm/s, suggesting fetal anaemia. This was confirmed on fetal blood sampling, but recovery was suggested with a reticulocyte count of 16.8%. The fetal karyotype was normal, 46,XY. Fetal IgM was positive for Parvovirus. A week later, severe fetal anaemia was suspected and intrauterine transfusion carried out. Altogether three transfusions were given. At 31/40 weeks, the mother presented to her local hospital with suspected preterm labour, a caesarean section was carried out because of fetal compromise on cardiotocography. The baby was in poor condition at birth and resuscitation was stopped at 45 min of age. The post-mortem examination confirmed the hydrops and proved persistent Parvovirus infection, cardiac involvement and severe liver fibrosis.HPVB19 generally follows a benign course with intrauterine therapy; however, in this case, the fetus died despite successful transfusions. The reasons for this are discussed.

Fetal magnetic resonance imaging in prenatal diagnosis of central nervous system abnormalities: 3-year experience.

OBJECTIVE: To evaluate the comparative merits of ultrasound and fetal magnetic resonance imaging (MRI) in the correct antenatal diagnosis of suspected central nervous system abnormalities. METHODS: A retrospective review of 27 consecutive pregnancies referred for fetal MRI for suspected central nervous system abnormalities between July 1998 and July 2001. Women were referred for the MRI examination when further anatomical and/or pathological clarification of the ultrasound scan findings was needed. Antenatal ultrasound scan and MRI were reviewed in relation to the findings on postpartum investigations. RESULTS: Data were complete for 26 pregnancies. The median gestational age at the time of the ultrasound examination was 26 weeks (95% CI 24 weeks 2 days to 28 weeks 1 day). The median gestational age at the time of magnetic resonance imaging was 27 weeks' gestation (95% Cl 26 weeks 1 day to 29 weeks 2 days). Eight fetuses had associated skeletal, renal and/or cardiac abnormalities previously noted on ultrasound examination. MRI confirmed the ultrasound diagnosis in 15/26 cases (58%). It changed the diagnosis to the correct one in 7/26 (27%) and misdiagnosed four cases (15%). Three of the four cases that were misdiagnosed on MRI occurred in the first 18 months of our 36-month experience. CONCLUSION: Ultrasound remains the primary imaging modality for prenatal diagnosis. Fetal MRI appears to be a useful adjunct to ultrasound to confirm or exclude certain abnormalities; this will consequently help in the counselling of parents and assist in planning further management. However, like any imaging technique, the sensitivity and specificity of the test are likely to improve with experience.

Noninvasive techniques to detect fetal anemia due to red blood cell alloimmunization: a systematic review.

OBJECTIVE: To estimate, in maternal red blood cell alloimmunization, the diagnostic value of fetal ultrasonography and Doppler blood flow velocity in the evaluation and prediction of fetal anemia. METHODS: Literature from 1970 to 2000 was identified using general bibliographic databases (MEDLINE and EMBASE), the Cochrane Library and relevant specialist register of the Cochrane Collaboration, and by checking reference lists of known primary and review articles. Studies were selected if the accuracy of the fetal ultrasound parameters or Doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard (fetal hemoglobin). The diagnostic tests evaluated were ultrasound measurement of the fetal spleen perimeter and Doppler studies of blood velocity estimates in the umbilical vein, ductus venosus, middle cerebral artery, thoracic aorta, and umbilical vessel combined with the thoracic aorta. Study selection, quality assessment, and data abstraction were performed independently and in duplicate. Data from the selected studies were abstracted as 2 x 2 tables comparing the diagnostic test result with the reference standard. Diagnostic accuracy was expressed as likelihood ratios. RESULTS: The review included eight primary studies with 362 pregnancies affected by red cell alloimmunization. Prospective patient recruitment and complete population details were reported in half of the selected studies (four of eight). Only one study reported masking the diagnostic test results to clinicians. The diagnostic test performance varied widely according to the type of the test evaluated and the cutoff level used to define fetal anemia, which varied from study to study. The diagnostic test study of highest methodological quality reported a positive likelihood ratio of 8.45 (95% confidence interval 4.69, 15.56) and negative likelihood ratio of 0.02 (95% confidence interval 0.001, 0.25) for maximum middle cerebral artery Doppler velocity. CONCLUSION: The literature reporting noninvasive techniques to predict fetal anemia is methodologically poor and a standard approach to the evaluation of these techniques is lacking. A recommendation for practice cannot be generated without further rigorous research.

Etiology and outcome of hydrops fetalis.

OBJECTIVES: To identify the etiology and pregnancy outcome of hydrops fetalis in a cohort of pregnancies referred to a tertiary maternal fetal medicine center in the UK. These data allow the review of a large series of pregnancies affected by hydrops fetalis and emphasize the importance of investigation and then treatment of individual cases. This provides parents with improved information and especially specific prognostic information. METHODS: A retrospective review of 63 consecutive cases of hydrops fetalis managed between September 1996 and March 1999. RESULTS: Of the pregnancies, 12.7% (n = 8) were associated with an 'immune' etiology. Of these, 62.5% (n = 5) had fetal anemia due to anti-D, 25% (n = 2) anti-Kell and 12.5% (n = 1) anti-c antibodies. The remaining 55 cases (87.3%) had a non-immune cause. Eight (14.5%) were due to human parvovirus B19 infection. Fourteen cases (25.5%) were associated with aneuploidy and, in four (7.3%), a primary hydrothorax was the cause of the non-immune hydrops fetalis. A cardiac cause was found in five (9.1%) cases. Three of these had supraventricular tachycardia and one had congenital complete heart block. Cystic hygroma was associated with hydrops fetalis in six cases. Twin-twin transfusion syndrome was the cause for hydrops in two cases. Massive transplacental hemorrhage was identified in one case. Fetal akinesia and muscular dystrophy caused hydrops in one case each. In 14.5% (8/55) of cases no obvious cause was identified and these were classified as 'idiopathic'. Three other cases could not be classified because parents declined investigations (unclassified). In the pregnancies with non-immune hydrops fetalis, the outcome was favorable in 27.3% (15/55) of cases. CONCLUSION: The prognosis of hydrops fetalis differs markedly between different etiological groups. Etiologies range from treatable causes with a good outcome and probably no long-term side-effects (as in case of parvovirus B19), to others which are incompatible with life or are associated with considerable perinatal morbidity and mortality.

Renin gene expression in fetal kidneys of pregnancies complicated by twin-twin transfusion syndrome.

Twin-twin transfusion syndrome (TTTS) complicates one in five monochorionic pregnancies and is generally associated with high mortality and morbidity. One twin (the recipient) grows appropriately and has polyhydramnios while the other (the donor) may have a reduced growth velocity and severe oligohydramnios. The disparities in amniotic fluid volumes represent differences in fetal urine output. These differences occur secondary to hemodynamic changes, in which the vascular arrangement of placental anastomoses in TTTS leads to unidirectional flow from the donor to the recipient twin. A better understanding of the pathophysiology may contribute to improved management of this morbid condition. We studied three consecutive prospectively diagnosed stillborn twin pairs affected by early-onset TTTS. Renin gene expression was studied in sections of fetal kidneys with immunocytochemistry using a renin antiserum and with in situ hybridization using riboprobes complementary to renin mRNA, and renin-secreting cells (RCC) were counted. The overall maturation of the renal cortex was assessed by the percentage of immature glomeruli. The donor twin kidneys were smaller than those of the recipients, but the maturation of the renal cortex was not significantly different (28.2% immature glomeruli in the donor and 24.4% in the recipient kidney). The donor kidney showed increased renin gene expression with hyperplastic juxtaglomerular apparatuses (JGAs) that contained excess RCCs (median 20.02 [25th-75th centiles, 5.4, 25.1 RCCs per 100 glomeruli]). In contrast, the recipient kidney was virtually devoid of these cells (0.04 [0, 0.36] RCCs per 100 glomeruli; P < 0.05). In the donor kidney, increased renin release may, by a local action, contribute to renal vasoconstriction and oliguria. Increased renin and/or angiotensin II in the blood passing through the placental anastomoses may, by an endocrine action, suppress renin synthesis in the recipient kidney, thereby increasing renal blood flow and causing polyuria and polyhydramnios. These changes in the renal RAS could thus contribute to the pathogenesis of TTTS. The renal renin changes noted here may represent a contributory or compensating mechanism, the success of which may dictate the overall survival of the twin pregnancy and allow better understanding of the pathophysiology and perhaps therapy that may be employed in this condition.

Outcome after prenatal diagnosis of hypoplastic left-heart syndrome: a case series.

BACKGROUND: Traditionally, after prenatal diagnosis of hypoplastic left-heart syndrome (HLHS) couples have been offered termination of pregnancy or comfort care. Success of postnatal surgical options such as the Norwood procedure have been associated with survival of up to 60%. Whether survival is affected by the congenital anomaly being identified prenatally or postnatally remains uncertain. METHODS: We reviewed all cases of prenatally diagnosed HLHS referred to the Fetal Medicine Unit at Birmingham Women's Hospital over 6 years between 1994 and 1999. FINDINGS: 87 cases of HLHS were referred at a median gestational age (95% CI) of 23 (19-37) weeks. Of these, 53 (61%) chose prenatal karyotyping. The overall frequency of abnormal karyotype was found in seven of 59 cases (12%) and associated structural anomalies in 18 of 87 (21%). After counselling, 38 of 87 couples (44%) chose termination of pregnancy. Of the remaining 49 fetuses, 11 (23%) were not considered for postnatal surgery because of parental choice and they died after compassionate care. Of the 36 babies who had surgery postnatally, 12 survived (33%). We recorded a survival rate of 38% for the stage-1 Norwood procedure in the prenatally diagnosed HLHS in our centre. These data suggest that at the point of prenatal detection, the overall survival rate for fetuses with HLHS is 25% (if terminated pregnancies are excluded). INTERPRETATION: Fetal echocardiography allows early diagnosis of HLHS and gives clinicians the opportunity to triage this group dependent on prenatal findings, including karyotyping and the exclusion of other structural anomalies. These prospective data provide up-to-date information on the basis of which parents can make decisions.

Hepatocyte growth factor activator (HGF-A) and its zymogen in human placenta.

HGF-activator (HGF-A) is a circulating serine protease known to be responsible for activation of hepatocyte growth factor (HGF). Active HGF is thought to be an important regulator of trophoblast growth. In vitro, HGF-A is produced via proteolytic cleavage of its zymogen by thrombin. Immunocytochemistry and Western immunoblotting were performed using human placental tissue from all three trimesters with an antibody that recognizes both HGF-A and its zymogen. Western immunoblotting revealed a 97 kDa band equivalent to the zymogen in placenta from all three trimesters. A smaller 34 kDa band equivalent to HGF-A was only seen in first and second trimester placenta. The anti-HGF-A/zymogen antibody demonstrated immunostaining in placental villi and membranes throughout gestation. Within first trimester villi immunostaining was strongest within the syncytio- and cytotrophoblast layers, but was also seen within stromal and endothelial cells. Likewise, in third trimester placenta the syncytio-cytotrophoblast layer showed the strongest immunoreactivity. In vitro, HGF can induce trophoblast DNA synthesis and the localization of HGF-A to the peri-villous trophoblast layer (which expresses c-met, the HGF receptor) suggests that it may be responsible for activation of pro-HGF at this site. This adds further weight to the hypothesis that HGF in vivo is an important regulator of trophoblast growth.

The outcome of pregnancy in Kell alloimmunisation.

OBJECTIVE: To assess management and outcome of pregnancies with anti-Kell in the West Midlands in the UK over 13 years. DESIGN: A retrospective review of casenotes. SETTING: A regional referral clinic for red cell alloimmune disease and fetal medicine unit at a university hospital. POPULATION: Sixty-five pregnancies were identified in 52 Kell-sensitised women with Kell positive partners from the records of the Birmingham Blood Transfusion Centre. METHODS: Information from the casenotes was entered on a database and comparisons were made using the SPSS for Windows statistics package. MAIN OUTCOME MEASURES: Mode of sensitisation, degree of fetal or neonatal anaemia, need for transfusion, gestation at delivery, birthweight and pregnancy outcome. RESULTS: Alloimmunisation was transfusion-related in 29 pregnancies and pregnancy-induced in 33. The cause could not be identified in three cases. There were 22 proven Kell positive fetuses, of which 18 were affected, in which alloimmunisation was pregnancy-related in 12 cases and transfusion-related in five. Antibody titres and amniotic fluid OD450 were not helpful in management. Severe or very severe disease occurred in 50% of the affected pregnancies (9/18). There was no difference in pregnancy outcome between transfusion or pregnancy induced sensitisation. CONCLUSIONS: Anti-Kell alloimmunisation is an uncommon cause of serious anaemia in a significant proportion of affected pregnancies. There appears to be no difference between that caused by pregnancy or transfusion. Estimation of fetal haemoglobin concentration by cordocentesis is recommended, as antibody titres and amniocentesis are not helpful.

Prenatal diagnosis and outcome in sacrococcygeal teratomas: a review of cases between 1992 and 1998.

A review of sacrococcygeal teratomas diagnosed in the antenatal period in the West Midlands region over a six year interval is reported. The aim of the study was to assess the contribution of ultrasound scanning to the management of cases and to determine the outcome of prenatally diagnosed sacrococcygeal teratomas. A retrospective review of 10 cases was performed to obtain pregnancy details, ultrasound scan data and outcome information. Two fetuses were electively aborted. Perinatal mortality was 62.5% in the remaining cases with all stillbirths and neonatal deaths occurring in babies delivered preterm (at or before 34 weeks' gestation). Marked increase in tumour size (mainly vascular/solid) was observed in five of the fetuses, which was often associated with local compression effects and the development of hydrops. Eight out of 10 cases were delivered vaginally, one following aspiration of the large cystic tumour. Three of the four neonates surviving to surgery underwent successful resection of their benign tumours. As well as guiding prognosis, serial ultrasound scans may also allow the mode of delivery to be planned more effectively. The importance of a multidisciplinary team approach to these difficult cases is emphasized.

Unexpected rapid rise of maternal serum anti-D levels during pregnancy.

Two cases are described where hydrops faetalis developed as a result of very sudden and unexpected rises in serum anti-D levels. In both cases intravascular intrauterine transfusion was employed and a favourable outcome obtained. These cases show that continued vigilance is required even when anti-D levels are low. Weak antibody titres may be detected using enzyme-treated red cells, and failure to use this more sensitive technique meant that in one of the cases the initial weak antibody was not detected. There is currently a debate about the use of this more sensitive test in view of the increased work involved.

The 4 year outcome following the demonstration of bilateral renal pelvic dilatation on pre-natal renal ultrasound.

There is little in the literature regarding long-term prognosis in cases of fetal pyelectasis and calyceal dilatation. The aim of this study was to correlate antenatal ultrasound findings with outcome in a large group of children, most of whom had routine antenatal mid-trimester scans. 75 babies with bilateral pyelectasis and calyceal dilatation in the pre-natal period and complete radiological and clinical data were identified over a 3 year period. Pre-natal ultrasound was correlated with results of post-natal investigation and the frequency of post-natal surgery was established. Follow-up was documented to discharge or to at least 4 years of age. Prognosis was related to the degree of pelvic dilatation, but neonatal morbidity was much more likely to be associated with pre-natal calyceal dilatation and/or hydroureter. 68% (51 of 75) of babies had insignificant abnormalities on post-natal investigation, defined as either transient fetal pyelectasis and calyceal dilatation, extrarenal pelves, or transient neonatal pyelectasis and calyceal dilatation. Five babies died in the neonatal period, all classified as either moderate or severe disease. Of the surviving 70 cases (93.3%), 27% had renal anomalies that required treatment by prophylactic antibiotics or surgery. The remaining babies were conservatively managed and followed as outpatients. One child required transplantation and a further two had a severe degree of chronic renal failure by the age of 4 years. These data will be of value in prospective counselling.

A case of recurrent congenital fetal anomalies associated with a familial subtelomeric translocation.

The potential of a new fluorescent in situ hybridization technique is discussed, which uses a complete set of telomeric probes to reveal cryptic chromosome rearrangements that remain undetected by standard cytogenetic analysis. We report the obstetric history of a patient who had a termination of pregnancy at 20 weeks for a fetus with multiple congenital anomalies but a normal male karyotype using conventional G-banding analysis on a mid-trimester placental biopsy. In a subsequent pregnancy, a diaphragmatic hernia and intra-uterine growth restriction were detected at 34 weeks' gestation and a fetal blood sample showed a normal female karotype. However, her child was born with dysmorphic features and additional severe abnormalities including microcephaly, anophthalmos and left fixed talipes. The child has shown marked developmental delay. In view of a strong family history of congenital abnormalities and recurrent miscarriage suggestive of a familial translocation, a fluorescent in situ hybridization technique using specific telomeric probes was performed on blood from the affected child and her parents. An unbalanced subtelomeric translocation was detected involving the long arms of chromosomes 2 and 7 in the child and a balanced translocation was detected in her father. Accurate genetic counselling and the opportunity for early prenatal diagnosis can now be offered to this family.

Ontogeny of hepatocyte growth factor (HGF) and its receptor (c-met) in human placenta: reduced HGF expression in intrauterine growth restriction.

Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.

Fetal thyrotrophin: the best indicator of long term thyroid function after in utero exposure to iodine-131?

A woman treated with radioiodine for thyrotoxicosis was subsequently found to be 19 weeks pregnant at the time of treatment. Fetal thyroid hormone levels in utero were normal or mildly elevated but fetal thyrotrophin (TSH) levels were very high. The baby remained euthyroid after birth, but required treatment with thyroid hormone replacement to normalise the TSH level. Neurodevelopment was normal at age 36 months.

Bladder visualisation as a prognostic sign in oligohydramnios-polyhydramnios sequence in twin pregnancies treated using therapeutic amniocentesis.

Oligohydramnios-polyhydramnios sequence in twin pregnancies may be managed by aggressive amniocentesis and is described in nine consecutive cases. In four of the nine pregnancies both twins survived, one pair died in the neonatal period, and the other four pairs all suffered intrauterine death. The median number of amnioreductions performed was five (range 2-7). In this series the reaccumulation of urine in the bladder of the 'stuck twin' was a predictive prognostic marker of survival in both twins, with a sensitivity and specificity of 100%.

Prenatal diagnosis in the United Kingdom--an overview.

Prenatal diagnosis is now a well-established part of health care in the UK. Cytogenetic or molecular diagnostic analysis following amniocentesis, chorionic villus sampling or cordocentesis is in routine practice and identification of 'at risk' pregnancies using biochemical screening or ultrasound is widespread. Professional guidelines have been established covering both sampling procedures and diagnostic testing, and legislation is in place regarding termination of pregnancy and pre-implantation diagnosis. The close liaison of the various groups of professionals involved has led to well-developed prenatal diagnostic and screening services within the UK. These links have been the major contributory factor to the current state of prenatal diagnosis and have been of great benefit to patients undergoing prenatal testing.

Structural chromosome anomalies in congenital diaphragmatic hernia.

In order to determine the outcome and associated chromosomal and structural anomalies in fetuses diagnosed in utero as having a congenital diaphragmatic hernia, we reviewed 48 consecutive cases referred to our regional Fetal Diagnostic Unit between 1988 and 1995. All babies were delivered in units with appropriate neonatal resuscitation facilities. Thirteen babies [34 per cent of those tested, confidence interval (CI) 19-49 per cent] had karyotypic abnormalities. Three had trisomies but the other nine had more complex karyotypic abnormalities including translocations, deletions, and marker chromosomes. Twenty-one fetuses (44 per cent, CI 30-58 per cent) had additional ultrasound abnormalities which affected the heart in ten cases (21 per cent). Overall, 13 babies survived (27 per cent, CI 14-40 per cent). In babies with normal chromosomes and no additional structural abnormalities the survival rate was 50 per cent (CI 25-75 per cent). Poor outcome was not predicted by early gestation at diagnosis, the hernial contents, or the presence of polyhydramnios. We conclude that parents should be counselled about prognosis with information derived from series of prenatally diagnosed diaphragmatic hernias. The investigations offered should include a detailed ultrasound examination, particularly of the heart, and karyotyping by fetal blood sampling.