Life After Surviving Fontan Surgery: A Meta-Analysis of the Incidence and Predictors of Late Death.

AIM: We now know that 20-40% of patients with a single ventricle will develop heart failure after the second decade post-Fontan surgery. However, we remain unable to risk-stratify the cohort to identify patients at highest risk of late failure and death. We conducted a systematic review of all reported late outcomes for patients with a Fontan circulation to identify predictors of late death. METHODS: We searched MEDLINE, Embase and PubMed with subject terms ("single ventricle", "Hypoplastic left heart syndrome", "congenital heart defects" or "Fontan procedure") AND ("heart failure", "post-operative complications", "death", "cause of death", "transplantation" or "follow-up studies") for relevant studies between January 1990 and December 2015. Variables identified as significant predictors of late death on multivariate analysis were collated for meta-analysis. Survival data was extrapolated from Kaplan-Meier survival curves to generate a distribution-free summary survival curve. RESULTS: Thirty-four relevant publications were identified, with a total of 7536 patients included in the analysis. Mean follow-up duration was 114 months (range 24-269 months). There were 688 (11%) late deaths. Predominant causes of death were late Fontan failure (34%), sudden death (19%) and perioperative death (16%). Estimated mean survival at 5, 10 and 20 years post Fontan surgery were 95% (95%CI 93-96), 91% (95%CI 89-93) and 82% (95%CI 77-85). Significant predictors of late death include prolonged pleural effusions post Fontan surgery (HR1.18, 95%CI 1.09-1.29, p<0.001), protein losing enteropathy (HR2.19, 95%CI 1.69-2.84, p<0.001), increased ventricular end diastolic volume (HR1.03 per 10ml/BSA increase, 95%CI 1.02-1.05, p<0.001) and having a permanent pacemaker (HR12.63, 95%CI 6.17-25.86, p<0.001). CONCLUSIONS: Over 80% of patients who survive Fontan surgery will be alive at 20 years. Developing late sequelae including protein losing enteropathy, ventricular dysfunction or requiring a pacemaker predict a higher risk of late death.

Novel multiplex oligonucleotide-conjugated bead suspension array for rapid identification of enterovirus 71 subgenogroups.

A high-throughput multiplex bead suspension array was developed for the rapid subgenogrouping of EV71 strains, based on single nucleotide polymorphisms observed within the VP1 region with a high sensitivity as low as 1 PFU. Of 33 viral isolates and 55 clinical samples, all EV71 strains were successfully detected and correctly subgenogrouped.

Prevalence and predictors of premature discontinuation of dual antiplatelet therapy after drug-eluting stent implantation: importance of social factors in Asian patients.

AIM: Premature discontinuation of antiplatelet therapy is an independent predictor of late stent thrombosis. We sought to determine the prevalence and predictors of premature discontinuation of antiplatelet therapy after drug-eluting stent implantation among patients in Asia. METHODS: A total of 207 consecutive patients who underwent drug-eluting stent implantation at our institution was followed up after 1 year. Premature discontinuation of antiplatelet therapy was defined as omission of aspirin and/or clopidogrel for 1 week or more. RESULTS: Four (1.9%) patients died and the remaining 203 patients formed the study population. Prevalence of premature discontinuation of antiplatelet therapy was 12.8% (n= 26, aspirin, n= 12; clopidogrel, n= 9; both, n= 5). The median duration between stent implantation and discontinuation of antiplatelet therapy was 2.8 months. Reasons for discontinuation included cost (n= 1), gastric discomfort (n= 1), allergy (n= 3), bleeding (n= 3), advice from doctors (n= 7) and no reason (n= 11). Logistic regression showed that living alone was the only independent predictor of premature discontinuation of dual antiplatelet therapy (50.0% vs 11.3%, P= 0.001). CONCLUSION: Among Asian patients who have undergone drug-eluting stent implantation, 12.8% discontinued dual antiplatelet therapy within 12 months. Living alone is associated with a fivefold increase in risk of premature drug discontinuation.

Utilisation of emergency medical service among Singapore patients presenting with ST-segment elevation myocardial infarction: prevalence and impact on ischaemic time.

BACKGROUND: Previous studies in Western countries found that the emergency medical service (EMS) was under-used in patients with myocardial infarction. AIM: We sought to determine the prevalence of immediate EMS utilisation among Singapore patients presenting with ST-segment elevation myocardial infarction (STEMI), and correlated the use of the EMS with the symptom-to-balloon and door-to-balloon times. METHODS: We studied 252 patients admitted with STEMI to our institution from August 2008 to September 2009. Information regarding demographic characteristics, whether EMS was used, reperfusion procedural details and mortality rates were collected prospectively. RESULTS: Among the recruited patients, 89 (35.3%) used the EMS (EMS group) and 163 (64.7%) did not use the EMS (non-EMS group). In the latter group, 98 (60.1%) arrived at our institution through their own transport, 56 (34.4%) first consulted general practitioners, and 9 (5.5%) initially consulted another hospital without acute medical services. Among the 245 (out of 252, 97.2%) patients who received percutaneous coronary intervention (PCI), the EMS group was more likely to undergo primary PCI (P= 0.003) while the non-EMS group was more likely to undergo non-urgent PCI (P= 0.002). In patients who underwent primary PCI, the EMS group had a shorter symptom-to-balloon time (average difference 81.6 min, P= 0.002). The door-to-balloon time was similar for both groups. CONCLUSION: Despite the availability of a centralised EMS, 64.7% of patients with STEMI did not contact EMS at presentation. These patients were less likely to receive primary PCI and had a significantly longer symptom-to-balloon time.

Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6.

A hybrid approach for fusing 4D-MRI temporal information with 3D-CT for the study of lung and lung tumor motion.

PURPOSE: Accurate visualization of lung motion is important in many clinical applications, such as radiotherapy of lung cancer. Advancement in imaging modalities [e.g., computed tomography (CT) and MRI] has allowed dynamic imaging of lung and lung tumor motion. However, each imaging modality has its advantages and disadvantages. The study presented in this paper aims at generating synthetic 4D-CT dataset for lung cancer patients by combining both continuous three-dimensional (3D) motion captured by 4D-MRI and the high spatial resolution captured by CT using the authors' proposed approach. METHODS: A novel hybrid approach based on deformable image registration (DIR) and finite element method simulation was developed to fuse a static 3D-CT volume (acquired under breath-hold) and the 3D motion information extracted from 4D-MRI dataset, creating a synthetic 4D-CT dataset. RESULTS: The study focuses on imaging of lung and lung tumor. Comparing the synthetic 4D-CT dataset with the acquired 4D-CT dataset of six lung cancer patients based on 420 landmarks, accurate results (average error <2 mm) were achieved using the authors' proposed approach. Their hybrid approach achieved a 40% error reduction (based on landmarks assessment) over using only DIR techniques. CONCLUSIONS: The synthetic 4D-CT dataset generated has high spatial resolution, has excellent lung details, and is able to show movement of lung and lung tumor over multiple breathing cycles.

IS1394 from Pseudomonas alcaligenes N.C.I.B. 9867: identification and characterization of a member of the IS30 family of insertion elements.

A new insertion sequence designated IS1394 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X) by entrapment in plasmid pUCD800 which carries the Bacillus subtilis sacB and sacR genes. The 1100-bp sequence contains 27-bp inverted repeats with 4 bp mismatch and has one long open reading frame, spanning 92.1% of the entire IS. The deduced 338 amino-acid sequence demonstrated homology (varying from 65% to 78% similarity and 36-67% identity) to transposases encoded by the IS30 family of IS elements. Comparison of four different IS-sacB junction sequences showed that IS1394 generated 3-bp direct repeats of target DNA upon insertion. IS1394 is present in at least 10 copies in the P25X genome but none was detected in its endogenous plasmid pRA2. Hybridization experiments revealed that the distribution of IS1394 is limited to closely related strains, being present in three copies in Pseudomonas putida NCIB 9869 (P35X) and two copies in Pseudomonas alcaligenes ATCC type strain (ATCC 14904).

Cloning and sequences of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putida P35X.

Pseudomonas putida P35X (NCIB 9869) metabolises phenol and cresols via a chromosomally encoded meta-cleavage pathway. A 13.4-kb fragment of the chromosome involved in encoding phenol catabolism was cloned and characterized. Deletion analysis and nucleotide sequencing of a 6589-bp region, in conjunction with enzyme assays, were used to identify the phhKLMNOP genes encoding the phenol hydroxylase, the phhB gene encoding catechol 2,3-dioxygenase (EC 1.13.11.2) and the phhQ gene that encodes a small ferredoxin-like protein. The genes are organised in an operon-like structure, in the order phhKLMNOPQB, and the deduced amino-acid sequences share high homology (68.3-99.7%) with those of the plasmid-encoded genes dmpKLMNOPQB of Pseudomonas sp. strain CF600. Genetic evidence is presented that the difference in the growth substrate ranges of Pseudomonas P35X and CF600 are due to the effector activation specificities of the regulators of these systems, rather than the substrate specificities of the catabolic enzymes.

Characterization of IS1474, an insertion sequence of the IS21 family isolated from Pseudomonas alcaligenes NCIB 9867.

A new insertion sequence (IS) designated IS1474 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X). IS1474 is a 2632 bp element which showed a characteristic IS structure with 12 bp inverted repeats (IRs) flanking a 2608 bp central region. IS1474 contained four open reading frames (ORF1-ORF4), two in each orientation. Similarities were detected between ORF1 and ORF2 and the putative transposases of the IS21 family. Sequences upstream from IS1474 were found to display up to 89% homology with IS53 from Pseudomonas syringae suggesting that IS1474 had inserted into another related IS element designated IS1475. An open reading frame, ORF5, located at the junction of IS1474 and IS1475, showed similarities with the IstB protein of IS21 and could possibly be the transposase subunit of IS1475. Transposition assays showed that IS1474 transposed at a relatively low frequency leading to cointegration with target plasmids. Hybridization studies showed that IS1474 is present in at least 13 copies in the chromosome of P25X and one copy on its endogenous plasmid.

Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region.

The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1.

Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867.

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.

Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes.

Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::XIn hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.

Subtyping of Neisseria gonorrhoeae auxotype-serovar groups by pulsed-field gel electrophoresis.

Genomic DNA from 48 Neisseria gonorrhoeae isolates was digested with low-frequency cleavage (LFC) endonucleases (SpeI and NheI) and analysed by contour-clamped homogeneous electric fields electrophoresis (CHEF). The restriction patterns generated were reproducible, stable, easy to read and offer a more practical approach than restriction endonuclease analysis (REA) with high-frequency cleavage (HFC) endonucleases. Strains sharing common auxotypes and serovars could be differentiated and correlation with auxotype/serovar (A/S) classes was demonstrated for some, but not all strains. The strains were distributed into 18 A/S classes and 38 SpeI and 40 NheI restriction patterns. This greater discriminatory power of CHEF REA should allow subdivision of strains within common A/S classes.

Differentiation of Neisseria gonorrhoeae IB-3 and IB-7 serovars by direct sequencing of protein IB gene and pulsed-field gel electrophoresis.

Serotyping of Neisseria gonorrhoeae based on the differential recognition by a panel of six monoclonal antibodies (MAbs) against Protein IB (PIB) is a valuable tool for studying the epidemiology of gonorrhoea. However, the predominance of certain serovars in specific geographic regions necessitates the development of new MAbs or new genotyping approaches. Nucleotide and amino-acid sequence analysis of PIB from strains within the IB-3 and IB-7 serovars revealed strain differences within the same serovar. Based on the generation of PIB nucleotide and deduced amino-acid sequences that centred on amino-acid residues 196 and 237, eight serovar IB-3 strains and nine serovar IB-7 strains were separately subdivided into five groups. Intra-serovar differences were also established by pulsed-field gel electrophoresis (PFGE) of macro-restriction fragments generated by SpeI- and NheI-cleavage of genomic DNA. There was good correlation between the results based on PIB gene PCR-sequencing and those based on PFGE analysis of macro-restriction fragment patterns. These data demonstrate the high precision of PFGE analysis and indicate that this approach can be used as a rapid epidemiological subtyping system for major serovars of N. gonorrhoeae.

Plasmid profiles compared with serotyping and pyocin typing for epidemiological surveillance of Pseudomonas aeruginosa.

The plasmid profiles of 112 clinical isolates of Pseudomonas aeruginosa were determined by two reproducible, rapid, plasmid screening methods. Plasmid DNA was present in 15% of isolates examined. Plasmids varied in size from 1.2 X 10(6) to 60.2 X 10(6) mol. wt. The dominant serotypes encountered were O:11 and O:4, which comprised 38% and 12% of strains, respectively. Four pyocin types (1, 10, 3 and 5) dominated (respective frequencies: 56, 15, 12 and 6%). Reproducibility of pyocin typing was distinctly inferior to both plasmid profiling and serotyping. Strains of identical serotypes could be further differentiated by dissimilar plasmid profiles. Serologically untypable or polyagglutinable strains were successfully characterised by plasmid profile patterns. Thus, plasmid profiling was shown to be a useful adjunct to serotyping for the epidemiological typing of P. aeruginosa.

RT-PCR, nucleotide, amino acid and phylogenetic analyses of enterovirus type 71 strains from Asia.

A specific and sensitive method based on RT-PCR was developed to detect enterovirus 71 (EV71) from patients with hand, foot and mouth disease, myocarditis, aseptic meningitis and acute flaccid paralysis. RT-PCR primers from conserved parts of the VP1 capsid gene were designed on the basis of good correlation with sequences of EV71 strains. These primers successfully amplified 44 strains of EV71 including 34 strains isolated from Singapore in 1997 and 1998, eight strains from Malaysia isolated in 1997 and 1998, one Japanese strain and the neurovirulent strain EV71/7423/MS/87. RT-PCR of 30 strains of other enteroviruses including coxsackievirus A and B, and echoviruses failed to give any positive amplicons. Hence, RT-PCR with these primers showed 100% correlation with serotyping. Direct sequencing of the RT-PCR products of 20 EV71 strains revealed a distinct cluster with two major subgroups, thus enabling genetic typing of the viruses. The genetic heterogeneity of these strains culminated in amino acid substitutions within the VP1, VP2 and VP3 regions. The sequencing of a 2.9 kb fragment comprising the capsid region and the major part of 5' UTR of two Singapore strains revealed that they belonged to a group distinct from the prototype EV71/BrCr strain and the EV71/7423/MS/87 strain. The dendrogram generated from 341 bp fragments within the VP1 region revealed that the strains of Singapore, Malaysia and Taiwan belong to two entirely different EV71 genogroups, distinct from the three genogroups identified in another recent study.

Pulsed-field gel electrophoresis for differentiation of hospital isolates of Klebsiella pneumoniae.

Restriction enzyme analysis by pulsed-field gel electrophoresis (PFGE) was developed for differentiation of hospital isolates of Klebsiella pneumoniae. Restriction patterns generated by SpeI digestion of genomic DNAs of 36 isolates from patients in two major teaching hospitals established 34 PFGE types. All strains were typable by this technique and the SpeI restriction patterns were reproducible, stable and easy to interpret. As PFGE profiles generated were heterogenous, the incidence of cross-infection appeared to be low in each of the hospitals. The higher discriminatory power of PFGE when compared to conventional restriction endonuclease analysis (REA) suggests that this technique will be very useful for epidemiological investigations of nosocomial K. pneumoniae outbreaks.

An institutional outbreak of Salmonella enteritidis in Singapore.

A large outbreak of food poisoning occurred in Singapore in March 1995 when a total of 188 inmates in an institution was taken ill. Salmonella enteritidis was isolated from the stool cultures of 35 inmates (16 symptomatic and 19 asymptomatic). All the isolates were of the serotype profile 0:1, 9, 12 and H:g, m (antigen phase I); all were sensitive to ampicillin, ceftriaxone, chloramphenicol, co-trimoxazole and ciprofloxacin. Plasmid profile analysis and restriction enzyme fragmentation patterns (REFPs), as generated with EcoRI and HindIII, of a 60 kb plasmid obtained from these isolates were all identical, confirming that the outbreak resulted from a single source of infection. Stratified statistical analysis of food-specific attack rates strongly implicated imported canned luncheon pork consumed by the inmates on 26 March 95 as the single most probable cause of the food poisoning [p < 10(6), Mantel-Haenszel weighted odds ratio (OR) = 14.33; 95% confidence interval (CI) = 6.20-33.15]. The median incubation period of this outbreak was 19.3 hours and the median duration of illness was three days. The outbreak was rapidly brought under control through prompt implementation of epidemic control measures which comprised active search for diarrheal cases, rectal swabbing of asymptomatic inmates, isolation of those found to be infected, and maintenance of a high standard of personal, food and environmental hygiene.

A spatiotemporal-based scheme for efficient registration-based segmentation of thoracic 4-D MRI.

Dynamic three-dimensional (3-D) (four-dimensional, 4-D) magnetic resonance (MR) imaging is gaining importance in the study of pulmonary motion for respiratory diseases and pulmonary tumor motion for radiotherapy. To perform quantitative analysis using 4-D MR images, segmentation of anatomical structures such as the lung and pulmonary tumor is required. Manual segmentation of entire thoracic 4-D MRI data that typically contains many 3-D volumes acquired over several breathing cycles is extremely tedious, time consuming, and suffers high user variability. This requires the development of new automated segmentation schemes for 4-D MRI data segmentation. Registration-based segmentation technique that uses automatic registration methods for segmentation has been shown to be an accurate method to segment structures for 4-D data series. However, directly applying registration-based segmentation to segment 4-D MRI series lacks efficiency. Here we propose an automated 4-D registration-based segmentation scheme that is based on spatiotemporal information for the segmentation of thoracic 4-D MR lung images. The proposed scheme saved up to 95% of computation amount while achieving comparable accurate segmentations compared to directly applying registration-based segmentation to 4-D dataset. The scheme facilitates rapid 3-D/4-D visualization of the lung and tumor motion and potentially the tracking of tumor during radiation delivery.