Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA.

Bacterial specific primers were used to amplify 23S rRNA genes from a representative strain from each of the 23 serogroups of the pathogenic Leptospira interrogans and 8 strains from 6 serogroups of the non-pathogenic Leptospira biflexa. Only regions of extreme variability, which had been identified on the basis of homology-based search of all the 23S rRNA sequences available in GenBank database, were sequenced from the amplified products. PCR primers that had the potential to distinguish L. interrogans from L. biflexa species were designed from the derived sequences and a sensitive PCR protocol developed. The PCR method enabled the differentiation of the 59 strains of the 23 serogroups of L. interrogans from the 8 strains of 6 serogroups of L. biflexa. Further investigation by 16S rDNA sequencing of two strains of L. interrogans, which gave unexpected PCR results, provided evidence that they had been misclassified and hence we propose to reassign them to L. biflexa.

Rapid distinction between Leptonema and Leptospira by PCR amplification of 16S-23S ribosomal DNA spacer.

The PCR amplification of the genomic DNA of Leptonema illini strain 3055 using primers directed against conserved regions of the rRNA operon provided evidence that the 16S and 23S rRNA genes were linked via an intergenic spacer region. The sequencing of the intergenic spacer region indicated that it was 435 nucleotides in length and sequence similarity searches revealed that it bore no homology to any known sequences including tRNA available in databases. Further investigations using Southern blot hybridization revealed that there were two copies of these linked genes in the genome. However, similar PCR studies on a representative strain from each of the 23 serogroups of Leptospira interrogans, which are pathogenic, and eight strains from the 6 serogroups of Leptospira biflexa, which are non-pathogenic, revealed that the 16S and 23S rRNA genes were not linked.

Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans.

Based on (i) an analysis of Leptospira 16S rDNA sequences determined by us and of those from databases and (ii) a previously published finding that restriction fragment length polymorphisms (RFLPs) within the Leptospira 16S and 23S rDNA were detected by nine restriction enzymes and these RFLPs allowed categorisation of Leptospira into eight genospecies, we predicted that one particular DdeI restriction site polymorphism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans. Two PCR-based methods, namely allele-specific amplification (ASA) and PCR-RFLP, were tested for the rapid detection of the DdeI restriction site polymorphism. One or two representative strains from each of nine genospecies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were tested by PCR-RFLP. Our experiments showed that the ASA method was not as specific as intended, but the PCR-RFLP method was useful for rapid identifications of the genospecies interrogans. We have not only confirmed a previous finding and extended the number of samples particularly from the genospecies biflexa, weilii, and inadai, but also simplified a previous PCR-RFLP protocol.

Bladder reconstruction in rabbits with glutaraldehyde-stabilized amniotic membranes.

Glutaraldehyde-treated human amniotic membranes were used to repair rabbit bladders after supratrigonal cystectomies. The membranes maintained the integrity of the bladders until healing and reepithelialization occurred. There was no significant loss of bladder capacity or decreased renal function postoperatively. Calcification did not occur on the membranes but was noted on chromic sutures retaining the membranes in 7 of 27 bladders. These findings suggest that glutaraldehyde-stabilized amnion is well tolerated by the urothelium and may serve as a suitable material for replacement of genitourinary tissues.

A prospective comparison of two expanded polytetrafluoroethylene grafts for linear forearm hemodialysis access: does the manufacturer matter?

BACKGROUND: The function and patency of standard 6-mm Goretex (W.L. Gore and Associates, Flagstaff, AZ) and Impra (Impra, Inc., Tempe, AZ) expanded polytetrafluoroethylene (e-PTFE) grafts for hemodialysis as radial-antecubital linear arteriovenous fistulae for dialysis are compared. STUDY DESIGN: A randomized clinical trial was conducted in two community dialysis centers and in one hospital-based center serviced by one vascular surgical practice, that performed the access surgery. Selection of linear forearm access, as opposed to other hemodialysis graft configurations, was at the discretion of the surgeon. Candidates for linear grafts had palpable radial pulses with a normal Allen test and normal digital Doppler flow in the hand. Linear grafts were placed using end-to-side anastomoses to the artery and vein, and the graft type was determined by randomization. Primary patency was determined by first episode of thrombosis, first revision, or angioplasty of the graft. Secondary patency after thrombectomy, revision, or angioplasty was determined when the graft was no longer clinically usable, and a new graft needed to be placed as a parallel conduit in the forearm or in another site. Statistical analysis was by actuarial life-table methods. RESULTS: There were 131 linear forearm grafts in 117 patients. The Impra and Goretex groups were equally matched for gender and major risk factors, except for smoking, which was more common in the Goretex group. Minimum followup was 24 months. Life table primary patencies at 1 year (Impra 43%, Goretex 47%) and at 2 years (Impra 30%, Goretex 26%) were not statistically different (p = 0.78); secondary patency was also equal at 1 year (Impra 49%, Goretex 69%) and at 2 years (Impra 33%, Goretex 41%) (p = 0.15). Discontinuance of use of a patent graft, complications, episodes of thrombosis, and the need to replace the original graft occurred in the two groups without a statistically significant difference. CONCLUSIONS: In the linear forearm position from the radial artery to an antecubital vein, there is no difference in the performance of 6-mm standard e-PTFE grafts on the basis of manufacturer, whether Goretex or Impra. On the basis of performance, linear forearm dialysis grafts are an acceptable method for hemodialysis access.

Ultrasonography of post-traumatic soft-tissue lesions.

This article highlights the varied uses of ultrasound for detection of trauma to soft tissues. The emphasis is on the diagnosis of tendon and muscle disruptions. Detection of soft-tissue foreign bodies by ultrasound, an important recent innovation, is also reviewed. Miscellaneous potential applications of ultrasound for diagnosing meniscal injuries, cartilage integrity, epidermoid cysts, and traumatic vascular malformations are discussed briefly. Ultrasound is efficacious because of its low cost, noninvasiveness, wide availability, and accuracy.

Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product.

Sequence analysis of 16S rRNA genes extracted from nucleic acids databases enabled the identification of a Leptospira biflexa (L. biflexa) signature sequence, against which a reverse primer designated L613, was designed. This primer, when used in conjunction with a universal bacterial specific forward primer designated Fd1, enabled the development of a LightCycler-based PCR protocol in which fluorescence emission due to binding of SYBR Green I dye to amplified products could be detected and monitored. A melting temperature (Tm), determined from the melting curve of the amplified product immediately following the termination of thermal cycling, confirmed that the product was that of L. biflexa. Agarose gel electrophoresis therefore was not necessary for identification of PCR products. The PCR protocol was very rapid, and consisted of 30 cycles with a duration of 20 s for each cycle with the monitoring of the melting curve requiring an additional 3 min. The whole protocol was completed in less than 20 min. The PCR protocol was also specific and enabled the identification of 18 strains of L. biflexa, whilst excluding 14 strains of L. interrogans and Leptonema illini. Two examples of its utility in improving work flow of a Leptospira reference laboratory are presented in this article. The use of a simple boiling method for extraction of DNA from all the members of the Leptospiraceae family DNA further simplifies the procedure and makes its use conducive to diagnostic laboratories.

Identification of Leptospira inadai by continuous monitoring of fluorescence during rapid cycle PCR.

Seven new Leptospira isolates from rats, a buffalo, and contaminated media showed either reactive serology against more than 1 serogroup or no reactive serology against a reference panel of 22 serovars in the microscopic agglutination test (MAT). Because of these inconclusive results, the 16S rDNA sequences of these isolates were determined and found to resemble that of the type strain of Leptospira inadai (L. inadai), serovar lyme strain 10, which is considered to be nonpathogenic for humans. Comparative analyses of other Leptospira 16S rDNA sequences from databases revealed a L. inadai-specific signature sequence, against which an amplification primer was designed. This primer when used in conjunction with an universal primer enabled the trial of a rapid PCR protocol in which fluorescence emissions due to binding of SYBR Green I dye to PCR products were continuously monitored during rapid thermal cycling. A melting curve acquired immediately after PCR was used to distinguish the intended product. The thermal cycling and continuous monitoring of fluorescence emission were accomplished by the LightCycler; the whole procedure of 30 PCR cycles and melting curve acquisition required only 20 minutes. The primer achieved the required specificity, as the intended PCR product resulted only from 6 confirmed L. inadai reference strains and 7 field isolates that had been verified as L. inadai by the 16S rDNA sequencing, but not from 16 reference strains of Leptospira belonging to 7 other genospecies. Furthermore, these experiments showed that the PCR protocol was robust because target DNA of different conditions, which were extracted by either 1 of the 4 methods used, could be detected.

Evaluating painful osteopenia in the elderly.

Osteopenia is a frequent finding on radiographs of elderly patients. When the exam is performed for skeletal pain, this finding may be significant. The differential diagnosis for osteopenia is extensive, but individualizing the patient work-up begins with a careful clinical history and laboratory studies. Appropriate radiographic exams can then be requested. A plain radiograph is always the starting point, followed by--as indicated--a nuclear bone scan, computed tomography, and magnetic resonance imaging.

Fibrous dysplasia: magnetic resonance imaging appearance at 1.5 tesla.

Fibrous dysplasia has been described in a small number of cases in the literature as showing low signal intensity on T1- and T2-weighted magnetic resonance images. We reviewed magnetic resonance scans of 13 patients with fibrous dysplasia to determine if there might be a characteristic appearance. All lesions had sharply defined borders and were of intermediate signal intensity on T1-weighted images. With T2 weighting, six lesions (46%) showed high signal intensity, four (31%) showed persistent intermediate signal intensity, and three (23%) showed mixed intermediate and high signal intensity. Ten lesions (77%) had inhomogeneous signal intensity and three (23%) had homogeneous signal intensity. We concluded that fibrous dysplasia does not have a characteristic appearance on magnetic resonance imaging. However, magnetic resonance may be helpful in establishing the diagnosis of fibrous dysplasia if low to intermediate signal intensity is seen on both T1- and T2-weighted images. This situation occurred in 54% of our cases, whereas the other 46% had nonspecific signal characteristics indistinguishable from many other bone lesions.

Reservoir hosts of Leptospira inadai in India.

Isolation of Leptospira from the kidneys of Rattus rattus wroughtoni hinton, Rattus rattus rufescens, Bandicota bengalensis and Bandicota indica was attempted in Bangalore in southern India. In total, 296 spirochaetes were isolated from 1,348 kidney cultures (an isolation rate of 22%). A batch of fifty-six isolates from India was identified, based on serological and polymerase chain reaction analysis, of which twenty-three isolates were identified as L. inadai by the World Health Organization/Food and Agriculture Organization Collaborating Centre for Reference and Research on Leptospirosis, in Brisbane. This is the first record of isolation of L. inadai from rodents. The preponderance of L. inadai in four different species of rodents suggests that these animals could be the natural reservoir hosts of L. inadai, and raises a critical question as to the likely impact of this species of Leptospira on the renal carrier status of other Leptospira pathogenic to humans and animals in this part of India. Virulence studies conducted at the University of Trieste in Italy, revealed that isolates of L. inadai from India were moderately or totally serum resistant when subjected to a serum killing test. To establish the possible seroprevalence of this species in the population, the inclusion of L. inadai in the battery of leptospiral antigens used for sero-epidemiological studies is recommended.

Leptospira interrogans antibodies in feral pigs from New South Wales.

The sera of 195 hunter-killed feral pigs (Sus scrofa), collected in New South Wales (Australia) from April to November 1995, were screened against a reference panel of 14 Leptospira interrogans serovars using a microscopic agglutination test (MAT). The panel represented those serovars previously isolated from wild and domestic mammals in mainland Australia. Antileptospiral agglutinins were detected in 20% of the sera tested and included nine L. interrogans serovars. The majority of serological reactors (63%) were to L. interrogans serovar pomona. Sera from 26% of immunoreactors cross reacted with antigens from one or more serovars. No differences were noted in the prevalence of L. interrogans antibodies between the sexes, or between pigs from areas of low and high rainfall. The implications of leptospirosis in feral pigs on the transmission of leptospires to wildlife, livestock, and humans are discussed.

Prevalence of antibodies of Leptospira serovars in beef cattle in central Queensland.

OBJECTIVE: To obtain up-to-date data on the prevalence of antibodies to Leptospira serovars in central Queensland beef herds preliminary to assessing their role in bovine subfertility and the role of cattle as a zoonotic reservoir. DESIGN: Sera from 2857 female cattle in 68 central Queensland beef herds were tested for antibodies to 14 Leptospira serovars using the microscopic agglutination test. Vaccination use and age of cattle were collected to enable the calculation of crude and age-stratified seroprevalences. RESULTS: The most commonly detected antibodies were to serovars hardjo (15.8% crude seroprevalence), tarassovi (13.9%), pomona (4.0%) and szwajizak (2.2%). Vaccinates were omitted from the hardjo and pomona seroprevalence data. The seroprevalence for hardjo and pomona tended to increase with age of the animals. CONCLUSION: These results are broadly similar to those of previous serological surveys. The data suggest that serovars other than hardjo, pomona and tarassovi, are unlikely to have a significant role in bovine subfertility and that cattle are unlikely to be a source of human infection with them in central Queensland.

Anteroinferior tears of the glenoid labrum: fat-suppressed fast spin-echo T2 versus gradient-recalled echo MR images.

OBJECTIVE: To compare fat-suppressed fast spin-echo (FSE) T2-weighted images with gradient-recalled echo (GRE) T2*-weighted images in the evaluation of anteroinferior labral tears. DESIGN: MR images were retrospectively reviewed by two radiologists masked to the history and arthroscopic findings. They separately interpreted the anteroinferior labrum as torn or intact, first on one pulse sequence and then, 4 weeks later, on the other sequence. The MR interpretations were correlated with the arthroscopic findings. PATIENTS: Nine patients with anteroinferior labral tears, and nine similarly-aged patients with normal, labra were studied. RESULTS AND CONCLUSIONS: Observer 1 had a sensitivity of 0.56 on the GRE images and 0.67 on the FSE images (P > 0.5), with a specificity of 1.0 for both sequences. Observer 2 had a sensitivity of 0.78 and a specificity of 0.89 for both sequences. In this small study there is no significant difference between GRE and fat-suppressed FSE images in their ability to diagnose anteroinferior labral tears. When evaluating the labrum with conventional MRI, axial fat-suppressed FSE T2-weighted images can be used in place of GRE images without a loss of accuracy.

Magnetic resonance imaging of myositis ossificans: analysis of seven cases.

Myositis ossificans typically presents as soft tissue swelling with progressive ossification on radiographs. Since magnetic resonance imaging (MRI) is commonly used to evaluate soft tissue masses, we analyzed eight MR examinations in seven patients with myositis ossificans to determine if typical patterns were present. One acute lesion had homogeneous intermediate signal intensity on T1-weighted images and high signal intensity on T2-weighted images. Two subacute lesions had low signal intensity margins with slightly increased signal intensity centers on T1-weighted images and very high signal intensity on T2-weighted images. Five chronic lesions had two different patterns. All five were well-defined with low signal intensity borders. Three had signal intensity patterns characteristic of fat on T1-weighted and T2-weighted images. The other two lesions had intermediate signal intensity on T1-weighted images and slightly increased signal intensity on T2-weighted images. We conclude that typical MR appearances of myositis ossificans do exist. A low signal intensity rim is a common finding. However, these patterns are not unique to myositis ossificans and resemble those that have been reported in other lesions. It is important to be aware of the spectrum of MR findings of myositis ossificans when considering the differential diagnosis of a soft tissue mass.

MR diagnosis of meniscal tears: analysis of causes of errors.

OBJECTIVE: MR imaging of the knee is an accurate method for diagnosing meniscal tears. However, MR findings do not always agree with surgical findings. In a retrospective study, we analyzed the various causes of incorrect MR diagnoses. MATERIALS AND METHODS: We reviewed a series of 400 MR examinations for suspected meniscal tears in which the diagnostic accuracy was 90%. In this group, we found 70 patients in whom the original MR diagnosis did not agree with the surgical findings. Three musculoskeletal radiologists independently reviewed each of the 70 MR examinations without knowledge of the original interpretation or the surgical findings. Their interpretations and the MR images then were correlated with the surgical findings. The original incorrect diagnoses were categorized as being due to unavoidable errors, errors in interpretation, or errors made because of equivocal MR findings of a tear. Unavoidable errors were defined as false-positive and false-negative diagnoses that could not be avoided, even in retrospect. RESULTS: Of the 83 original diagnostic errors made in the MR evaluation of 800 menisci, 33 (40%) were unavoidable errors, 32 (39%) were due to equivocal MR findings, and 18 (21%) were due to interpretation errors. The unavoidable errors consisted of 21 missed meniscal tears and 12 false-positive MR diagnoses. In the false-positive cases, the menisci were interpreted as torn on MR images by all three observers, but no tear was found at arthroscopy. Subtle MR findings that were equivocal for a tear caused both false-positive and false-negative diagnoses. Seven of the 18 interpretation errors occurred when normal variants were mistaken for a tear. CONCLUSION: Using conventional coronal and sagittal spin-echo MR imaging, we could not identify 21 (6%) of the 333 meniscal tears, even in retrospect. In addition, subtle findings that are equivocal for a tear may still make MR diagnosis of every torn meniscus difficult even for experienced radiologists. Unavoidable false-positive diagnoses due to healed tears or tears missed at arthroscopy are an infrequent problem occurring in only 1.5% of the original 800 menisci evaluated with MR imaging.