An analysis of the V1 and V2 regions of Vibrio cholerae and Vibrio mimicus 16S rRNA.

The V1 and V2 variable regions of the 16S rRNA gene of three strains of V. cholerae and one strain of V. mimicus were amplified by PCR. Fragments containing both regions were cloned into M13mp18 using Smal and sequenced by the dideoxy method. The 263-bp sequence from a strain isolated during the 1991 cholera outbreak in Brazil was deposited in Genbank under the accession number L05178. Except for an extra G in one of the strains, the three V. cholerae sequences were identical. The V. mimicus sequence was very similar, with only two substitutions. We compared these sequences with the Vibrio 16S rRNA sequences described by Dorsch et al. in 1992. It was noted that the V1 region, including helix 6 and its associated loop, comprised two different sizes and sequences in the various Vibrio species. While V. cholerae, V. mimicus, V. vulnificus, V. anguillarum and V. diazotrophicus had a 46-nucleotide V1, other species such as V. parahaemolyticus, V. proteolyticus, V. alginolyticus, V. campbellii and V. hollisae had longer 54- or 55-nucleotide regions, with a different consensus sequence. The phylogeny of Vibrio was analysed using the sequenced region and its equivalent in other species, by means of the "Phylip" software package. Species with a short helix 6 were grouped together, as were species with a long helix. Dorsh et al.'s analysis is discussed in relation to this "helix 6 split".

The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints.

Pathogenic Vibrio cholerae strains were compared by fingerprinting with arbitrarily primed polymerase chain reaction (AP-PCR). They were O1 classical and El Tor strains and recent non-O1 Bengal strains. Ten oligonucleotides from a total of fifty-two tested gave distinctive patterns, and these strains were separated into four groups. A second technique, amplification of 16S/23S rRNA spacers with a pair of oligonucleotides, was also used. Various bands were obtained, and the result can be treated as an additional fingerprint with a different pattern for each of the groups. The method of AP-PCR fingerprinting is fast and sensitive. A test of the stability of the El Tor patterns was done with a set of strains isolated during the present Brazilian epidemics. Examples of AP-PCRs with non-O1 strains are given. A typing scheme is proposed in which oligo 1 is first used, and depending on the fingerprint obtained, additional oligonucleotides are used to confirm the classification of the strain. It is proposed that the AP-PCR technique be used for epidemiological studies, analysing strains reaching new locations or environmental isolates suspected of being pathogenic. It will be particularly helpful in cases in which traditional methods cannot clearly classify the strain.

A different approach for the total correction of tricuspid atresia.

A new surgical procedure has been used successfully to correct tricuspid atresia in a 9-year-old girl. An external conduit containing a porcine aortic valve was positioned to lead from the right atrium to the underdeveloped right ventricle. The right ventricle was reconstructed with a large Dacron patch, thus providing a large cavity for the small hypoplastic right ventricle. The atrial and ventricular septal defects were closed. The patient made a satisfactory recovery and is doing well four months after the operation.

Focal infection of aortofemoral prostheses.

A canine model of unilateral aortofemoral bypass was utilized to study periprosthetic infections in woven, knitted, and velour Dacron vascular prostheses. These studies suggest that physical configuration, porosity, and time of inoculation all are important variables when managing an infected prosthesis, although size of inoculum seems less important.

Toxin-co-regulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence of a third allele of pilin gene.

Polymerase chain reaction has been used to detect the presence of the virulence associated gene, tcpA and part of the promoter distal region of the toxin-co-regulated pilus cluster in non-O1, non-toxigenic, Vibrio cholerae. The amplified regions were characterised by restriction fragment length polymorphism and heteroduplex motility assay. We describe the nucleotide sequence of the tcpA gene fragment from non-toxigenic vibrios from clinical and environmental sources. The present study shows that there are at least three types of the tcpA gene among V. cholerae and the primers specific for the classical tcpA gene, amplify all biotypes. A sequence similarity in other regions of the toxin-co-regulated pilus cluster is suggested. The evidences for the presence of this cluster among non-toxigenic vibrios is, to our knowledge, reported for the first time. The use of restriction fragment length polymorphism for typing the tcpA and studying the alleles distribution is proposed.

Detection of Vibrio cholerae and V. mimicus heat-stable toxin gene sequence by PCR.

Previously the heat-stable enterotoxin in Vibrio cholerae and V. mimicus has been detected by suckling mouse assay, a non-specific approach, and by DNA probes, a time-consuming method. This report describes a polymerase chain reaction (PCR) procedure for the detection of the stn (NAG-ST) and sto (O1-ST) gene sequences that is rapid and specific, allowing toxin gene molecular characterisation. A total of 34 V. cholerae and V. mimicus isolates was examined for ST and CT genes. The NAG-ST gene sequence was amplified in 13 of 22 non-O1/non-O139 V. cholerae and three of five V. mimicus strains. A new enterotoxin gene sequence pattern was found with MseI and TaqI restriction endonuclease PCR fragment digestion of two V. cholerae isolates, in addition to the pattern anticipated from the Genbank sequence, and found with the other ST+. These results show that ST-PCR detection is useful for the characterisation of V. cholerae and V. mimicus.

Extraanatomical bypass of the aortic root: an experimental technique.

Endocarditis involving a prosthetic aortic valve is associated with persistently positive blood cultures and aortic regurgitation. With rare exception, it is a fatal disease. An experimental technique was developed that would allow for removal of the infected aortic prosthesis with debridement and permanent closure of the aortic root. An extraanatomical outflow for the left ventricle was created using a valve-containing conduit between the apex of the left ventricle and the descending thoracic aorta (apicoaortic anastomosis). The procedure was performed on 5 mongrel dogs through a left thoracotomy without use of cardiopulmonary bypass. Four survived the procedure and required no cardiotonic support. One died as a result of a technical problem. Intraoperative pressure determination revealed a 0 to 15 mm Hg gradient across the apioaortic prosthesis and a left ventricular end-diastolic pressure of 0 to 4 mm Hg. Both postoperative angiocardiogram and postmortem examination confirmed patency of the aortocoronary bypass grafts and good function of the prosthesis.

Enzyme markers for Vibrio cholerae: identification of classical, El Tor and environmental strains.

Enzyme electrophoretic variants were studied in 49 strains of Vibrio cholerae using zymovar analysis. The following seven enzymes were selected for use: alanine dehydrogenase (ADH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucosephosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH). The results indicated the presence of three main groups defined chiefly by their GPI and 6PGDH variants. The first group, defined by possessing the variants GPI-2 and 6PGDH-3, contained all the 01 serovar and E1T or biovar isolates from cholera cases. The second group, defined by possessing the variants GPI-3 and 6PGDH-2, contained all the 01 serovar and classical biovar isolates; the third group was heterogeneous and included the 01 serovar isolates from environmental sources as well as isolates of other serovars (the so called NAGs, non-agglutinable with 01 antisera or NCVs). It is thus now possible to separate the epidemic strains of 01 serovar from other members of this serovar isolated from the environment. Zymovar analysis deals with differences which are a direct expression of the genome and seems to be unaffected by gross phenotypic changes such as smooth-rough variation and phage resistance. It is a promising tool for investigating bacteriological and epidemiological questions, in particular the significance of an environmental reservoir of cholera.

Identification of Vibrio cholerae by enzyme electrophoresis.

Zymovar analysis of 260 strains of Vibrio cholerae plus 3 reference strains of V. mimicus, using 13 structural loci, led to the grouping of strains in 73 zymovars (strain or group of strains sharing the same alleles). Effective separation of strains, distinction of V. cholerae strains from closely related V. mimicus and the detection of 2 vibrio strains, including one with two O1 serovars, in supposedly pure collection cultures, illustrate the potential of zymovar analysis in the identification of V. cholerae isolates. Two El Tor strains from USA, one CT+ and the other CT-, shared the same zymovar 71, while 127 typical El Tor strains belonged to zymovar 14.

A zymovar analysis of Vibrio cholerae isolated in Australia.

Zymovar analysis was used to study 50 strains of Vibrio cholerae O1 and 40 strains of V. cholerae non-O1 isolated in Australia. The strains were assigned to 42 zymovars; the O1 strains to 9 types and the non-O1 strains to 33 types, with no overlapping between serovars. All the human O1 isolates, regardless of their ability to produce cholera toxin (CT), and all the CT-producing O1 environmental isolates, were type Z14. The remaining O1 strains and the non-O1 strains belonged to a variety of zymovars, and more than one zymovar was present in some rivers.

Aortic reconstruction with crimped bovine pericardial conduits.

BACKGROUND AND AIM OF THE STUDY: A bovine pericardial conduit processed in glutaraldehyde was designed, incorporating the principle of crimping used for synthetic vascular prostheses. The crimping process did not affect the integrity of collagen fibers and tissue structure. This conduit, designed for aortic reconstruction, is available in different sizes, with or without a biological valve. METHODS: Between October 1989 and May 1997, 40 patients with aortic dissection, aortic aneurysm, aortic coarctation or aortoiliac occlusive disease underwent aortic reconstruction using this vascular substitute. Procedures included total reconstruction of the ascending aorta and aortic valve with reimplantation of coronary arteries (nine patients), single ascending thoracic aorta (six), descending thoracic aorta (two), aortic arch (one) and thoracoabdominal aorta (one); the abdominal aorta was reconstructed in 21 cases, including those undergoing aortoiliac or aortofemoral bypass. RESULTS: The hospital mortality rate was 20% (eight patients); causes of deaths were low cardiac output, recurrence of aortic dissection, multiple organ failure and bleeding. Mean follow up was 3.6 years; total follow up was 114 patient-years. Late conduit-related complications occurred in four patients, including a limb obstruction in one patient subjected to aortofemoral bypass and infection of three resulting in pseudoaneurysm (incidence of 3.5 +/- 1.8% per patient-year). All underwent reoperation. There were four late deaths due to sudden death, coronary artery disease, pneumonia and metabolic complications of diabetes and renal failure (incidence of 3.5 +/- 1.8% per patient year). The eight-year actuarial survival rate was 63.7 +/- 11.6%, including hospital mortality, and the eight-year actuarial freedom from conduit failure due to primary tissue structural degeneration was 100%. CONCLUSIONS: The crimping design provides a circular tube which makes construction of the anastomosis easier, retains its shape with bending, and avoids kinking. The material is very soft, easy to handle and suture, coapts nicely to suture lines resulting in a hemostatic anastomosis. The eight-year follow up demonstrated a satisfactory performance without report of fibrosis, calcification or aneurysmal dilation.

Early experience with crimped bovine pericardial conduit for arterial reconstruction.

A bovine pericardial conduit was developed in the laboratory incorporating the principle of crimping used for synthetic vascular prostheses. The pericardium was processed in glutaraldehyde and the tube was crimped by a technique which preserves the integrity of collagen fibres. This vascular substitute presents a non-thrombogenic and non-porous inner surface which does not require preclotting and does not leak. The material is very soft, easy to handle and suture, coapts nicely to suture lines resulting in a hemostatic anastomosis. The crimping design provides longitudinal elasticity and resistance to collapsing, retains its shape with bending and avoids kinking. Crimping provides a circular tube which makes the construction of the anastomosis easier. Experimental studies in dogs demonstrated absence of thromboembolism with the conduit implanted in the abdominal aorta. Fibrin accumulation was not noted in the convexities of the crimps. This conduit was designed for aortic and pulmonary reconstruction and available in different sizes with or without a biological valve. Initial clinical experience included its use in 10 patients with aortic dissections or aortic aneurysms from August 1989 to March 1990. A reconstruction of the abdominal aorta was performed in 2 patients, the descending thoracic aorta in 2, the ascending aorta in 2 and the ascending aorta including the aortic valve and reimplantation of coronary arteries in 4. For the latter 4, composite crimped pericardial tubes containing a porcine bioprosthesis were used. An additional patient with a single ventricle underwent a Fontan type operation also employing a valved crimped pericardial conduit.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac valve replacement with glutaraldehyde preserved aortic allografts.

From September 1984 to December 1988, 144 patients underwent cardiac valve replacement using glutaraldehyde preserved stent mounted aortic allografts. The mean age was 21.4 years (54.9% were 15 years old or younger). The mitral valve was replaced in 125 patients, the aortic valve in 7, the pulmonary valve in 3, the tricuspid in 1, the mitral and tricuspid in 1, and the aortic and mitral in 7. Hospital mortality was 2.8% (4 patients). Total follow-up was 396.3 patient-years and the mean follow-up was 2.8 years per patient. The 5-year actuarial survival was 73.8% +/- 11.9%. The 4-year actuarial survival for patients aged 15 or younger was 81.4% +/- 7.1%. The overall mortality was 2.8% +/- 0.8%/per patient-year. The incidence of valve-related complications was 4.8% +/- 1.1%/per patient-year, and the calcification rate was 3.0% +/- 0.9%/per patient-year and was the main complication reported in 12 patients, all under the age of 15 years. It occurred 14-47 months after implantation (mean 32.7 months). Five-year actuarial freedom from valve dysfunction due to calcification was 82.6% +/- 5.0% and for patients aged 15 or younger was 69.9% +/- 8.8%. The incidence of reoperation was 3.3% +/- 0.9%/per patient-year. These initial results demonstrate a 5-year actuarial freedom from primary valve failure due to fibrocalcification superior to the results obtained with xenobioprostheses in the paediatric age group.

Mitral valve replacement with glutaraldehyde preserved aortic allografts.

OBJECTIVE: To present long-term results after mitral valve replacement with stent mounted glutaraldehyde preserved aortic allografts in patients older than 15 years. The clinical support for this study was to combine the glutaraldehyde technique of biological tissue preservation with the advantages of allografts when compared to xenografts. This was demonstrated in previous studies using other methods of tissue processing. METHODS: Between September 1984 and November 1994, 70 patients aged 16-77 years (mean 35.4 years) underwent mitral valve replacement with this preserved and mounted allograft. Of these, 40 patients (57.2%) were aged 16-35 years and 15 (21.4%) were 20 years old or younger; 46 (65.7%) were females and 24 (34.3%) males. Single mitral valve replacement was performed in 60 patients and 10 were also subjected to other combined cardiac procedures. Human aortic valves were obtained during routine autopsy, processed in glutaraldehyde and mounted into flexible stents, using the same technique as that used for porcine bioprostheses. RESULTS: Hospital mortality was 1.4%. Total follow-up was 543.1 patient-years, corresponding to a mean follow-up of 7.9 years per patient. Echocardiography demonstrated a hemodynamic performance similar to porcine bioprostheses. Late mortality was 0.7 +/- 0.6% per patient-year and the causes were congestive heart failure in 2, prosthetic endocarditis in 1 and acute myocardial infarction in 1. The 12-year actuarial survival was 92.4 +/- 3.2%. The incidence of late complications was 5.2 +/- 1.2% per patient-year, including congestive heart failure, prosthetic endocarditis, periprosthetic leak, thromboembolic episodes, recurrence of rheumatic disease, coronary artery disease and allograft failure. Complications related to heart disease represented 2.8 +/- 0.6% and allobioprosthesis-related 2.4 +/- 0.5% per patient-year. The 12-year actuarial freedom from primary valve failure was 81.0 +/- 15.0%. The incidence of reoperations was 1.5 +/- 0.8% per patient-year and the main indication was prosthetic endocarditis. Other causes were periprosthetic leak, aortic insufficiency in the native aortic valve and allobioprosthesis dysfunction. Functional results demonstrated a significant improvement in patients clinical condition. CONCLUSION: This 12-year follow-up shows a very low incidence of primary allograft failure for patients older than 15 years undergoing mitral valve replacement, and much superior than our results with porcine bioprosthesis in the same age group. This supports our assumption that this investigational valve represents a new advance in cardiac valve surgery.