Outcome after preterm delivery of infants antenatally diagnosed with congenital heart disease.

OBJECTIVE: To determine outcome of delivery before 36 weeks gestation in babies diagnosed antenatally with serious congenital heart disease (CHD). STUDY DESIGN: A retrospective database review at 2 tertiary care fetal cardiology centers. Details of neonatal course and outcome were obtained for those antenatally diagnosed with serious CHD who were live born before 36 weeks gestation. RESULTS: Between January 1998 and December 2002, 9918 women were referred for fetal echocardiography. Serious CHD was diagnosed in 1191 fetuses (12%), of which 46 (4%) delivered prematurely. Median gestation was 33 (range 24-35) weeks, and median birth weight 1.56 (0.50-3.59) kg. Extracardiac/karyotypic anomalies occurred in 23 (50%). Twenty-six babies (57%) underwent neonatal surgery: 16 a cardiac procedure, 5 a general surgical procedure, and 5 both. Eight died during or after operation (31%). Two babies underwent interventional heart catheterization; both died. The overall mortality rate was 72%. Extracardiac/karyotypic anomalies increased the relative risk of death by a factor of 1.36. Mean hospital stay for those surviving to initial discharge was 46 (2-137) days. CONCLUSIONS: There is a very high morbidity and mortality rate in this group, particularly for those with extracardiac/karyotypic anomalies. This should be reflected in decisions over elective preterm delivery and when counseling parents.

Echocardiographic assessment of conjoined twins.

OBJECTIVE: To determine the accuracy of prenatal and postnatal echocardiography in delineating the degree of cardiac fusion, intracardiac anatomy (ICA), and ventricular function of 23 sets of conjoined twins with thoracic level fusion presenting to a single centre over a 20 year period. METHODS: 13 thoracopagus, 5 thoraco-omphalopagus, and 5 parapagus pairs presenting to the authors' institution between 1985 and 2004 inclusive were assessed. Echocardiographic data were analysed together with operative intervention and outcome. Twins were classified according to the degree of cardiac fusion: separate hearts and pericardium (group A, n = 5), separate hearts and common pericardium (group B, n = 7), fused atria and separate ventricles (group C, n = 2), and fused atria and ventricles (group D, n = 9). RESULTS: The degree of cardiac fusion was correctly diagnosed in all but one set. ICA was correctly diagnosed in all cases, although the antenatal diagnosis was revised postnatally in three cases. Abnormal ICA was found in one twin only in two group A pairs, one group B pair, and both group C pairs. All group D twins had abnormal anatomy. Ventricular function was good in all twins scanned prenatally, and postnatally function correlated well with clinical condition. Thirteen sets of twins in groups A-C were surgically separated; 16 of 26 survived. None from groups C or D survived. CONCLUSIONS: Prenatal and postnatal echocardiography accurately delineates cardiac fusion, ICA, and ventricular function in the majority of twins with thoracic level fusion. It is integral in assessing feasibility of separation. The outcome in twins with fused hearts remains dismal.

Interventional cardiac catheterisation in congenital heart disease.

As a result of recent technological advances, more types of congenital heart disease are amenable to treatment in the cardiac catheter laboratory than ever before.1 Improved imaging techniques allow for better selection of patients, and the development of a wide range of devices specifically for use in children means that many patients can avoid surgery altogether, while those with complex congenital heart disease may require fewer or less complex surgical procedures.2 This allows for a quicker recovery and a shorter hospital stay, and gives many patients an improved quality of life in the short to medium term. However, the long term outcome for many of the newer forms of intervention is still unknown.

Acute myocardial infarction as a cause of death in palliated hypoplastic left heart syndrome.

A 20 month old child with hypoplastic left heart syndrome died suddenly from a massive myocardial infarction 15 months after a hemi-Fontan operation. This was confirmed at postmortem examination and histological examinations. The sites of surgical reconstruction were all in good condition, there were no gross anatomical coronary abnormalities, and the coronary ostia were unobstructed. On microscopy the internal coronary arteries had notable intimal and medial thickening with narrowing of the lumen, although no thrombotic occlusion was seen. To the authors' knowledge, this is the first published report of arteriosclerosis of the coronary arteries in hypoplastic left heart syndrome. It raises the question as to whether there may be a primary histological abnormality in some children with this condition or whether some mechanism of accelerated arteriosclerosis is at work.

Concordance for hypoplastic left heart syndrome in a monochorionic twin pregnancy.

The risk of structural heart disease is significantly higher in twin pregnancies than in singleton pregnancies, but the concordance rate has been found to be relatively low, even in monochorionic pregnancies. This is the first report of a monochorionic twin pregnancy concordant for hypoplastic left heart syndrome (HLHS), the diagnosis having been made by fetal echocardiography at 15 weeks' gestation. The findings were confirmed at necropsy at 17 weeks' gestation, following termination of pregnancy. Both twins had mitral and aortic atresia, with severely hypoplastic aortic arches. This report adds weight to there being a genetic component to the cause of HLHS in some cases and illustrates how the findings from early fetal echocardiography with postmortem follow up can help to extend the understanding of the aetiology of this condition.

Percutaneous retrieval of central venous catheter fragments.

Children with indwelling central venous catheters are at risk of embolisation of catheter fragments. Often their underlying condition means that they are poor candidates for surgical removal. We describe six children who underwent uncomplicated percutaneous transcatheter retrieval (and one who underwent percutaneous line tip repositioning), and suggest that this approach should be the treatment of choice.

Coil occlusion of systemic venous collaterals in hypoplastic left heart syndrome.

OBJECTIVE: To assess the frequency of systemic venous collaterals to the atria, which may cause desaturation, after stage II reconstructive surgery for hypoplastic left heart syndrome (HLHS) and to determine whether coil occlusion prevents the need for surgical ligation. DESIGN: Prospective interventional study. SETTING: Tertiary referral centre. PATIENTS: 27 children with HLHS undergoing cardiac catheterisation between October 1996 and February 2001. INTERVENTIONS: 19 children were catheterised prestage II, 1 poststage II, and 17 prestage III. Aortic oxygen saturation (SaAo) and pulmonary artery pressure (pPA) were recorded. Angiography was performed into the left internal jugular vein to look for venous collaterals. If present, they were occluded with Cook MReye coils. Angiography was repeated to confirm occlusion, and SaAo and pPA were remeasured. RESULTS: Collaterals were found in 7 of 27 children: 1 poststage II and 6 prestage III. These were occluded with 1-3 coils without complication. Mean (SE) SaAo before occlusion was 80.2 (2.1)% in those with collaterals compared with 88.7 (1.0)% in those without (p = 0.007). There was no difference in mean pPA between the two groups. After coil occlusion mean SaAo rose to 83.8 (1.8)% (p = 0.007) and mean pPA rose from 12.5 (1.5) to 14.5 (1.8) mm Hg (p = 0.02). None required surgical ligation. CONCLUSION: Angiography should be performed at catheterisation before stage II and III surgery for HLHS to exclude systemic venous collaterals. If present, they may be safely and effectively occluded with coils to improve saturation and prevent the need for subsequent surgical ligation.

Isolation of Tn916-like conjugal elements from swine lot effluent.

Isolates of Enterococcus faecalis obtained from a swine farrowing house outflow were examined for genetic elements similar to Tn916. Of the enterococci isolated, 71% were resistant to tetracycline. Among the tetracycline-resistant enterococci isolated from the outflow samples, approximately 34% were able to transfer the tetracycline resistance phenotype to Bacillus thuringiensis in cross-genus matings. The frequencies of transfer for 10 random isolates were comparable to those for transfer of Tn916 from E. faecalis to B. thuringiensis. In addition, these elements were shown to mobilize plasmid pC194 between Bacillus species, as did Tn916. Southern blot and polymerase chain reaction (PCR) analysis showed these elements share extensive structural homology with Tn916. The selected conjugal elements were capable of transfer to a Bacillus recipient in a soil environment. When the swine waste was introduced into the soil, the tetracycline resistant fecal enterococci levels rose from essentially undetectable levels to approximately 4 x 10(4) and remained at this level for 4 weeks. After six months, including one winter, levels had decreased to 5 x 10(3).

Analysis of the requirement for a pUB110 mob region during Tn916-dependent mobilization.

Tn916-dependent mobilization of nonconjugative plasmids pUB110 and its derivative pUB110Deltam was compared. Deleting a 787-bp fragment from the pUB110 mob region created plasmid pUB110Deltam. Deletion of the mob region of pUB110 rendered the plasmid nontransferable by the conjugative plasmids of Bacillus thuringiensis subsp. israelensis. During matings between Bacillus subtilis (Tn916) and B. thuringiensis subsp. israelensis, however, Tn916-dependent mobilization of plasmids pUB110 and pUB110Deltam was observed at a frequency of approximately 2 x 10(-6) transconjugants per donor. The results show that Tn916-mediated conjugal transfer of plasmids is a mob-independent event. Jaworski and Clewell (J. Bacteriol 177; 6644-6651) recently demonstrated the presence of an IncP-like nicking site in the oriT of Tn916. These data suggest that a IncP-like nickling site is essential for Tn916-mediated plasmid transfer.

Functional comparison of conjugative transposons Tn916 and Tn925.

During interspecies matings between Bacillus subtilis and Bacillus thuringiensis subsp. israelensis, transfer of conjugative transposon Tn916 was detected at a frequency of 1.1 x 10(-4) transconjugants per donor. Tn916-dependent transfer of plasmids pC194 and pE194 was detected at frequencies of 1.4 x 10(-5) and 3.2 x 10(-7) transconjugants per donor, respectively. Similar frequencies were obtained during parallel matings with otherwise isogenic strains that contain Tn925 instead of Tn916. Tn916- or Tn925-dependent transfer of plasmids pC194 or pUB110 from the recipient to the donor (retrotransfer) was not observed during inter- or intraspecies matings. Transposon-mediated plasmid transfer by Tn916 and Tn925 is a Rec independent event. Thus, the data from studies in which otherwise isogenic donor and recipient strains were used indicated that Tn916 and Tn925 are, from a functional point of view, much more similar than previously suggested.

Tetracycline enhances Tn916-mediated conjugal transfer.

Pregrowth of the donor on medium containing tetracycline increased conjugative transposition of Tn916 and the transposon-dependent mobilization of pC194 19- to 119-fold in matings between Bacillus subtilis and Bacillus thuringiensis subsp. israelensis. Tn916 and pC194 transferred independently under these conditions. When Enterococcus faecalis was the donor and B. thuringiensis subsp. israelensis the recipient, pregrowth in tetracycline increased the conjugative transposition frequency by approximately 15-fold. Tetracycline-enhanced conjugation appeared during matings as short as 3 h in length. Pregrowth in tetracycline did not enhance conjugation in Bacillus sphaericus x B. thuringiensis subsp. israelensis or B. thuringiensis subsp. israelensis x B. subtilis matings. Incorporation of tetracycline into the mating medium, at concentrations that did not inhibit growth of the B. thuringiensis subsp. israelensis recipient, resulted in conjugation frequencies similar to those obtained by pregrowth of the B. subtilis donors in antibiotic-containing medium. The data suggest stimulation of donor function by tetracycline.

Molecular cloning and characterization of Mycobacterium paratuberculosis promoters in Escherichia coli.

DNA fragments from Mycobacterium paratuberculosis were cloned in the promoter probe plasmid pKO1. Of 957 recombinant DNA clones, 24 induced synthesis of galactokinase (the reporter gene) when these plasmids were transformed into an Escherichia coli strain deficient for the enzyme. A DNA insert from one putative promoter-containing plasmid, designated pAG5, was sequenced and shown to contain, a characteristic RNA polymerase binding site, a probable ribosomal binding site and a putative open reading frame.

Plasmid profile analysis, phage typing, and antibiotic sensitivity of Salmonella dublin from clinical isolates in the United States.

One hundred clinical isolates of Salmonella choleraesuis subsp. choleraesuis serovar dublin (Salmonella dublin) were examined for phage sensitivity, antibiotic resistance patterns, and plasmid content. Computer analysis of the lysis patterns observed by using 27 typing phages divided the S. dublin isolates into 26 groups. One lytic pattern (Designated pattern 16) contained 52% of the isolates examined whereas 16 isolates had unique patterns, and nine patterns had fewer than ten members. Although 14 antibiotic resistance patterns were observed among the 100 isolates, 79% of the isolates grouped in three major patterns. Seven plasmid groups were identified and designated A-G based on the large plasmids found in the isolates. Of the 100 isolates, 28 contained the plasmid profile of Group A, 28 were Group B, 7 were Group C, 34 were Group D, and 1 isolate each was observed in Groups E, F, and G. The strong association between antibiotic resistance pattern and plasmid type suggest that the drug resistance genes are plasmid borne.

Analysis of restriction endonuclease fragment patterns of DNA from Mycobacterium paratuberculosis.

The DNA from 31 isolates and a reference strain of Mycobacterium paratuberculosis was digested individually with restriction endonucleases BstE II and Pst I. DNA fragments were separated by gel electrophoresis and analyzed. The isolates were from 23 American states, Argentina and Nova Scotia. Twenty-seven were isolated from cattle, two from goats and two from sheep. With the exception of one isolate from cattle, all had restriction endonuclease fragment patterns identical to the fragment patterns for the reference strain, M. paratuberculosis ATCC 19698T. These results confirm other reports and indicate that organisms identified as typical M. paratuberculosis isolates are genetically very similar. It may be possible to use restriction endonuclease analysis to differentiate isolates of M. paratuberculosis from other slowly growing mycobacteria. The genetic similarity also indicates that it may be possible to develop a diagnostic probe that is specific for M. paratuberculosis.

Tn916-dependent conjugal transfer of PC194 and PUB110 from Bacillus subtilis into Bacillus thuringiensis subsp. israelensis.

The Staphylococcus aureus plasmids pC194 and pUB110 were introduced into Bacillus thuringiensis subsp. israelensis by using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. Plasmid transfer occurred only when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor that contained both pC194 and pUB110 and Tn916; plasmid transfer was not observed in the absence of the transposon. B. thuringiensis transconjugants resistant to chloramphenicol (Cmr) and tetracycline (Tetr) were detected at a frequency of 1.96 x 10(-6) per recipient cell, whereas the Tetr phenotype, but not the Cmr, was observed at a frequency of 1.09 x 10(-4). The converse, Cmr but not Tetr, was observed at a frequency of 2.94 X 10(-5). The transfer of pUB110 from B. subtilis to B. thuringiensis subsp. israelensis was observed at a frequency of 3.0 x 10(-6) per recipient cell but concomitant transfer of pUB110 and Tn916 was not observed. Mobilization of plasmid pE194 was not observed under these conditions. Transconjugants were detected in filter matings only, not in broth. The Tn916 phenotype was maintained during serial passage of B. thuringiensis without selection, whereas the pC194 phenotype was not. Unlike pC194, however, pUB110 remained stable in B. thuringiensis during several passages through nonselective medium. Southern hybridization analysis demonstrated that Tn916 had inserted into several different sites on the B. thuringiensis chromosome and that pC194 and pUB110 were maintained as an autonomous plasmid.

Introduction of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis.

The conjugative Streptococcus faecalis transposon Tn916 was introduced into Bacillus thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline (Tetr) were detected at a frequency of approximately 7.0 X 10(-7) per recipient cell during filter matings, whereas transfer of Tn916 was not observed in broth matings. The Tetr phenotype in subsp. israelensis was stable in the absence of antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome but insertion into plasmid DNA was not observed. Movement of Tn916 was demonstrated when Tetr B. thuringiensis transconjugants were mated with isogenic recipients. Southern hybridizations, however, showed that the resulting Tetr isolates contained Tn916 junction fragments that were nearly identical to the donor, suggesting that this movement resulted from transfer of chromosomal DNA from donor to recipient or from a fusion of mating cells, rather than conjugative transposition of the Tn element.

Isolation and analysis of restriction endonuclease digestive patterns of chromosomal DNA from Mycobacterium paratuberculosis and other Mycobacterium species.

A relatively rapid and efficient method for the extraction of chromosomal DNA from Mycobacterium paratuberculosis and other mycobacteria was developed. Approximately 25 to 50 micrograms of DNA could be extracted from 100 mg (wet weight) of cells, which was sufficient to perform several restriction endonuclease analyses from a single preparation. The DNA from five Mycobacterium species, including four strains of M. paratuberculosis and four strains of M. avium, was analyzed by this method. Digestion with the restriction endonucleases BstEII and PstI yielded the most definitive restriction patterns. For some strains, the restriction endonuclease analysis results were in agreement with the current identification of these organisms. The two strains of M. avium serotype 2 had identical fragment patterns. Similarly, the two strains of M. avium complex serotype 6 had identical fragment patterns. The three mycobactin-dependent M. paratuberculosis strains were very similar, whereas the mycobactin-independent M. paratuberculosis strain was more similar to the M. avium serotype 2 strains. Although many more cultures would need to be evaluated to determine correct groupings, the results of this study demonstrated the potential of restriction enzyme analysis for the differentiation of slowly growing mycobacteria.

Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis.

Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.

The biotechnology of Bacillus thuringiensis.

One of the challenges in the application of biotechnology to pest control is the identification of agents found in nature which can be used effectively. Biotechnology offers the potential of developing pesticides based on such agents which will provide environmentally sound and economically feasible insect control alternatives. Such an agent, the insect pathogen Bacillus thuringiensis, is the subject of intense investigations in several laboratories. Insecticides which use the entomocidal properties of B. thuringiensis are currently produced and sold worldwide; new products are currently in the development stage. Herein, the biology and genetics of B. thuringiensis and the problems associated with current products are critically reviewed with respect to biotechnology. Moreover, the economic and regulatory implications of technologically advanced products are evaluated.