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.

Angiogenic therapy for the chronically ischemic lower limb in a rabbit model.

The purpose of this study was to evaluate the long-term effectiveness of basic fibroblast growth factor (bFGF) in achieving neovascularization following ischemia from arterial ligation and to determine an optimal dosage level. We used an Ameroid constrictor to produce progressive occlusion of the left femoral artery of rabbits. At 2 weeks, the rabbits were randomized to receive intravenous injection of vehicle (group A, n = 15); 3 microg/kg/day bFGF (group B, n = 12); 10 microg/kg/day bFGF (group C, n = 12); or 16 microg/kg/day bFGF (group D, n = 15) for 3 days. At 1 to 37 days after surgery, we assessed limb neovascularization by transcutaneous oximetry (TCPO(2)), angiography, heart rate, arterial pressure, peripheral vascular resistance (PRU), and muscle blood flow (MBF) during steady-state intra-arterial infusion of saline (basal), acetylcholine, papaverine, or serotonin under anesthesia and capillary density (cap/mm(2)) and capillary per muscle fiber ratio (cap/F). Groups B and C showed significantly greater change in TCPO(2) over time than groups A and D (P < 0.0001). Group D showed the lowest TCPO(2) values from days 14 to 37 and group C the highest. Groups B and C showed a higher number of vessels filled with contrast agent than groups A and D (P < 0.0001). Calf cap/mm(2) and cap/F were significantly higher in groups B and C than groups A and D (P < 0.0001). Calf basal MBF values were higher in groups B and C than in groups A and D, but were not statistically significant. Group D showed the highest level in basal PRU. There were no significant differences in heart rate or blood pressure among the groups. These results show (1) treatment with bFGF has no adverse hemodynamic effects, (2) bFGF enhances angiogenesis and circulation at moderate doses, and these effects persist at least several weeks, and (3) high doses of bFGF may inhibit angiogenesis and collateral circulation.

Leptospira autumnalis isolated from a human case from Avadi, India, and the serovar's predominance in local rat and bandicoot populations.

Leptospira were successfully isolated from the urine of an Indian patient who had been clinically diagnosed as having leptospirosis. In an attempt to determine the source of this infection, 28 rats (Rattus rattus) and 58 bandicoots (Bandicota bengalensis) living in the vicinity of the patient's home in Avadi, a suburban area of the city of Chennai (Madras), India, were then investigated. Each animal was checked for infection by microscopical examination of fresh and stained urine, serological analysis of serum, and the culture of urine and kidney samples. Direct, dark-field, observation of fresh urine samples and examination of urine samples after Fontana's silver staining were found to be the least sensitive of the tests used. The results of the serological microscopic agglutination test (MAT) indicated that four (14.3%) of the rats and nine (16.1%) of the bandicoots had significant agglutinins, predominantly for the serogroups icterohaemorrhagiae and autumnalis. Leptospira were isolated from at least one culture of samples from one rat and each of four bandicoots. Each of these rodent isolates and the human isolate were typed as Leptospira interrogans serovar autumnalis.

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.

Are oblique views needed for trauma radiography of the distal extremities?

OBJECTIVE: To our knowledge, few studies exist on the importance of the oblique view when radiography of the distal extremities is performed after acute trauma. Our prospective study aimed to determine whether the oblique view uniquely revealed abnormalities or clarified findings when it was obtained along with routine frontal and lateral radiographs. SUBJECTS AND METHODS: We prospectively interpreted 1461 consecutive radiographic examinations of the distal extremities in patients presenting with acute trauma to four family medicine clinics. The anatomic sites radiographed included the ankle, foot, toe, wrist, hand, finger, and thumb. Each study was interpreted and given a diagnostic certainty score using the lateral and posteroanterior or anteroposterior views only and then scored again with the oblique view added. RESULTS: The examinations included 421 with abnormal findings, 34 with equivocal findings, and 1006 with normal findings. The addition of the oblique view changed the interpretation in 70 (4.8%) of the 1461 examinations. Of these changed interpretations, 39 were changed from equivocal to either positive or negative, three from positive to negative, and 28 from negative to positive. Addition of the oblique view increased diagnostic confidence: The percentage of examinations scored as having probably normal, equivocal, and probably abnormal findings decreased from 13.9% with two views to 8.4% with three views (p < .0001). The oblique view was equally valuable in the ankle, foot, toe, wrist, hand, finger, and thumb. CONCLUSION: In the distal extremities, the oblique view uniquely reveals abnormalities and increases the confidence of the final radiographic diagnosis when the oblique view is interpreted along with frontal and lateral radiographs.

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.

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.

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.

Basic fibroblast growth factor stimulates angiogenesis in the hindlimb of hyperglycemic rats.

Angiogenic growth factors including basic fibroblast growth factor (bFGF) have therapeutic value for chronic ischemia in nondiabetic animals. However, angiogenic therapy for chronic ischemia in a background of diabetes remains unexplored. In the present study, we evaluated the effects of exogenous bFGF on angiogenesis in streptozotocin-induced diabetic rats with ischemic and nonischemic limbs. We produced ischemia of the left lower limb by excising the superficial femoral artery. At 2 weeks, the rats received an intramuscular injection of vehicle (group A), 0.3 microg bFGF/day (group B), or 1 microg bFGF/day (group C), daily for 2 weeks. At 4 weeks, we assessed limb angiogenesis by skeletal muscle capillary density (cap/mm2) and capillary per muscle fiber ratio (cap/F) counts. Group C had significantly higher mean levels compared to group A for calf capillary density (P < 0.0024) and capillary per muscle fiber ratio in both thigh (P < 0.0015) and calf (P < 0.0001). There was a trend toward increased mean capillary per muscle fiber ratio with increasing dose. This trend was significant in the calf (P < 0.0015) and just missed statistical significance in the thigh. There was a similar trend in calf capillary density. We conclude that exogenous bFGF enhances angiogenesis and, possibly, collateral circulation in ischemic limbs of diabetic rats.

Real-time homogeneous assay of rapid cycle polymerase chain reaction product for identification of Leptonema illini.

Partial 16S rDNA sequences of eight Leptospira-like field isolates that reacted weakly or not at all to microscope agglutination test were found to be similar to the 16S rDNA sequence of the nonpathogen Leptonema illini-type strain 3055. Comparison of these sequences with those of Leptospira 16S rDNA sequences revealed a Leptonema species signature sequence for which a forward amplification primer was designed. This primer was used in conjunction with a bacterial-specific 16S rDNA universal reverse primer for developing a LightCycler-based rapid PCR protocol in which fluorescence emission due to the binding of SYBR green I dye to the amplified products was continuously monitored. A melting temperature (T(m)) determined from the melting curve of the amplified product immediately after PCR confirmed that the product was of Leptonema. The protocol for 24 samples consisting of 30 PCR cycles and melting curve acquisitions required 30 min to complete and agarose gel electrophoresis of the PCR products was not necessary. The method was specific as PCR products were detected from the seven Leptonema reference strains and the eight field isolates that had been previously verified as Leptonema by 16S rDNA sequencing, but not from the two representative strains from each of the eight Leptospira genospecies tested.

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.

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.

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.

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.

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.

MR diagnosis of labral tears of the shoulder: value of T2*-weighted gradient-recalled echo images made in external rotation.

OBJECTIVE: Because several studies have shown that conventional MR imaging can fail to diagnose a significant percentage of labral tears, some authors have proposed obtaining T2*-weighted gradient-recalled echo images with the humerus in external rotation. The purpose of our study was to determine whether the diagnostic accuracy of detecting anteroinferior labral tears by MR imaging would be improved by adding a T2*-weighted gradient-recalled echo sequence with the humerus in external rotation. MATERIALS AND METHODS: The study included 24 patients for whom axial MR images of the shoulder were obtained with the humerus in both the neutral position and external rotation. Two observers interpreted the images made in the neutral position and then noted any change in their interpretations after viewing findings on the external-rotation images. MR results were correlated with surgical findings. At surgery, 14 anteroinferior labra were found to be torn and 10 were found to be intact. RESULTS: Both observers identified one patient for whom a surgically proved labral tear was seen only on the external-rotation images. The sensitivity increased from 0.43 to 0.50 (p = .35) for observer 1 and from 0.36 to 0.43 (p = .35) for observer 2. The specificity of 0.90 for both observers remained unchanged. The accuracy improved from 0.62 to 0.67 for observer 1 and from 0.55 to 0.62 for observer 2. CONCLUSION: The addition of external-rotation T2*-weighted gradient-recalled echo images to the MR examination for tears of the anteroinferior labrum leads to a small but statistically insignificant increase in diagnostic sensitivity. We conclude that the small increase in sensitivity does not justify the routine use of this sequence.

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.