'Leptorapide' - a one-step assay for rapid diagnosis of human leptospirosis.

SUMMARY: Leptospirosis is a globally important zoonotic infection caused by spirochaetes of the genus Leptospira. It is transmitted to humans by direct contact with infected animals or indirectly via contaminated water. It is mainly a problem of the resource-poor developing countries of the tropical and sub-tropical regions of the world but outbreaks due to an increase in travel and recreational activities have been reported in developed and more industrialized areas of the world. Current methods of diagnosis are costly, time-consuming and require the use of specialized laboratory equipment and personnel. The purpose of this paper is to report the validation of the 'Leptorapide®' test (Linnodee Ltd, Northern Ireland) for the diagnosis of human leptospirosis. It is a simple one-step latex agglutination assay performed using equal volumes of serum sample and antigen-bound latex beads. Evidence of leptospiral antibodies is determined within minutes. Agglutination is scored on a scale of 1-5 and the results interpreted using a score card provided with the kit. Validation has been performed with a large sample size obtained from individuals originating from various parts of the world including Brazil and India. The test has shown sensitivity and specificity values of 97·1% and 94·0%, respectively, relative to the microscopic agglutination test. The results demonstrate that Leptorapide offers a cost-effective and accurate alternative to the more historical methods of antibody detection.

Survival of leptospires in commercial blood culture systems revisited.

AIM: To assess the ability of commercial blood culture systems to maintain leptospires. METHODS: Nine different commercial blood culture bottles were compared for their ability to maintain four leptospiral strains at two temperatures, 30 degrees C and 37 degrees C. Bottles were subcultured at 48 hours, and one, two, three, and four week intervals and examined microscopically for the presence of viable leptospires. RESULTS: The results were comparable with those of an earlier study, which showed that different commercial blood culture systems varied in their ability to maintain leptospires. CONCLUSIONS: No single factor appears to influence the viability of leptospires in blood culture systems. In general, the combination of an aerobic blood culture and an incubation temperature of 30 degrees C enhances the viability of leptospires, and hence would increase the chances of their subsequent isolation from suspected cases of leptospiraemia.

An evaluation of three commercial kits for use as screening methods for the detection of leptospiral antibodies in the UK.

AIMS: To compare three commercial screening tests--the PanBio leptospiral IgM enzyme linked immunosorbent assay (ELISA), the Biolisa leptospiral IgM ELISA, and the indirect haemagglutination assay (IHA)--with the microscopic agglutination test (MAT) and two "in house" ELISAs--urease and horseradish peroxidase (HRP)--for the detection of leptospiral antibodies in a local UK and Eire population. METHOD: Two hundred sera submitted for a differential diagnosis of leptospirosis were tested by all methods. A further 142 sera from patients with antibodies to toxoplasma, Epstein-Barr virus (EBV), hepatitis A virus, rheumatoid factor, Borrelia burgdorferi, Mycoplasma pneumoniae, syphilis, cytomegalovirus, and Q fever were tested for crossreactivity. RESULTS: Compared with the MAT, sensitivity and specificity were found to be: PanBio, 90%/94%; Biolisa with sorbent, 100%/85%; and IHA, 54%/95%. Seven of 200 trial sera gave false negative results with PanBio; 14 of 200 trial sera gave false positive results with Biolisa with sorbent, as did a further 25 of the 142 sera tested for potential crossreactivity. Two of 142 sera gave crossreactions with PanBio and IHA (one each). CONCLUSIONS: The degree of false positivity seen with the Biolisa suggests that the recommended positive value of > or = 26 Eu/ml should be reassessed using pools of sera from local populations. When the cut off value was reassessed, using a value of > or = 40 Eu/ml, a sensitivity and specificity of 96% and 94%, respectively, was achieved. Even the modified Biolisa appears to be over sensitive and to show a high degree of non-specificity. The IHA, although specific (95%), lacked sensitivity in this study. The PanBio appeared to be the most suitable as a screening test for leptospiral IgM in the UK, although it would be advisable for all positive test results to be confirmed by a different enzyme immunoassay and the MAT.

Anticardiolipin antibodies in leptospirosis.

The clinical course and serology of 16 cases of leptospirosis in an area with an unusually high endemic infection rate were studied to gain further insight into the pathology of the secondary immune phase that is typical of the disease. IgG anticardiolipin antibody concentrations were measured by immunoassay and found to be increased in eight serologically confirmed cases with severe complicated disease, compared with eight patients with relatively uncomplicated leptospirosis who had IgG anticardiolipin concentrations within the control reference range. This previously unreported association suggests that leptospira may induce vascular endothelial injury in severe cases and expose crypt antigens or induce conformational change of cell surface phospholipids. Leptospirosis may provide a model for an infective origin of some cases of the antiphospholipid syndrome.

Comparison of the DIFCO and Patoc 1 slide antigens in the screening of leptospirosis.

A comparison of the Patoc 1 slide agglutination (SAT) and DIFCO slide agglutination test for the screening of leptospirosis in man, cattle and coypu (Myocastor coypus Molina) is reported. The economic costs, convenience and availability of the antigens for the tests are analysed. It is recommended that slide agglutination methods alone are not sufficient for the routine diagnosis of leptospirosis.

Serological study of leptospiral infections and endogenous uveitis among horses and ponies in the United Kingdom.

The prevalence of antibody titres to a range of 20 leptospira antigens in the serum of horses and ponies with no ophthalmic abnormalities and with ophthalmoscopic evidence of endogenous uveal inflammatory disease was determined using a microscopic agglutination technique. Titres against leptospira antigens were observed in 13 out of 138 (9.1 per cent) animals with no ophthalmic abnormalities, and in three out of 27 (11.1 per cent) animals with anterior uveitis. Serovar sejroe was common to all seropositive animals with anterior uveitis. The results show that leptospira infection is not a major factor in the aetiology of equine anterior uveitis in the UK, but suggests that the organism may be associated with some cases of recurrent anterior uveitis.

A comparative investigation and identification of Leptospira interrogans serogroup icterohaemorrhagiae strains by monoclonal antibody and DNA fingerprint analyses.

The identification of Leptospira interrogans icterohaemorrhagiae strains from a number of Reference Laboratories were confirmed using monoclonal antibody (MoAb) and DNA restriction endonuclease (EcoR1) analyses. With a few exceptions, strain fidelity was demonstrated. Three clinical isolates and one isolate from a rat (Rattus norvegicus) were identified on DNA fragment patterns and found to be similar to the reference strains, icterohaemorrhagiae copenhageni, I. "icterohaemorrhagiae" Ictero I and I. icterohaemorrhagiae RGA.

The activities of some metabolic enzymes in the intestines of germ-free and conventional chicks.

The metabolic enzymes alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in mucosal homogenates and these enzymes, together with glucose-6-phosphatase (EC 3.1.3.9), were measured in homogenates of isolated enterocytes from germ-free (GF) and conventional (CV) chicks which were either fed continuously until they were killed or were subjected to a 16 h fast before killing. The intestine of the GF chicks was generally lighter than that of the CV controls. The activity of alkaline phosphatase was greater in the mucosal homogenates of the CV chicks compared with the GF birds, but the concentrations of acid phosphatase and isocitric dehydrogenase were not different in the two groups. In the isolated enterocytes the concentration of all enzymes expressed per mg DNA, except alkaline phosphatase, was higher in the GF chicks. Expressed per mg protein there was no significant difference in enzyme activity in the two groups. Fasting caused a reduction in intestinal weight and total mucosal protein in both groups but the reduction was greater in the GF chicks compared with the CV controls. In the GF chicks, fasting caused a significant fall in acid phosphatase and isocitric dehydrogenase activities of the mucosal homogenate, whereas in the CV chicks only acid phosphatase fell to a significant extent. In the isolated enterocytes feeding caused a marked fall in protein per mg DNA in the CV chicks; fasting tended to reduce enzyme concentrations in the GF chicks but to have less effect in the CV group except for alkaline phosphatase where there was a marked rise in activity. It is suggested that the difference in enzyme activities in the mucosal homogenates and isolated enterocytes might resulted from (a) the presence of a much greater lamina propria in the CV compared with the GF chicks and (b) the greater mitotic activity in the fed CV chicks yielding a much larger number of smaller immature cells.

Activities of some metabolic enzymes in the small intestinal mucosa during pregnancy and lactation in the rat.

The food intake, gut weight, gut length, mucosal protein and mucosal activities of alkaline phosphate (EC 3.1.3.1), acid phosphate (EC 3.1.3.2), isocitric dehydrogenase (EC 1.1.1.42) and glucose-6-phosphate (EC 3.1.3.9) were measured in rats during pregnancy, lactation and after the young were weaned. In general, the quantities measured increased slightly during pregnancy and considerably during lactation, reaching maximum values during the 3rd weeks of lacation and falling more or less rapidly after the young were weaned to the same levels as those in unmated animals. However, the gut length and mucosal protein remained higher even 3 weeks after weaning, so that weight per unit length and specific enzyme activities (per mg protein) tended to be lower in mated than in unmated rats. Changes in the specific activities of enzymes indicate alterations of the metabolic function of the enterocytes during breeding similar to changes reported for digestive enzymes. It is suggested that the intestine may reflect changes that take place in the liver.

The absorption and secretion of calcium in the gastrointestinal tract of germ-free and conventional chicks.

1. The uptake transport and secretion of calcium into the gastrointestinal tract of germ-free (GF) and conventional (CV) chicks was determined by a variety of in vivo and in vitro techniques using 45Ca as a marker following a standard fast-fed regimen. 2. Following an oral dose of Ca in conscious chicks, the transit time of digesta in the GF birds was appreciably faster than in the CV controls. 3. Although the proportion of the Ca dose absorbed was similar in both groups of chicks, the accumulation of Ca in the intestinal wall was much greater in CV birds. Distribution of the labelled Ca in plasma and bone was similar in both groups. 4. Following intravenous CA injection in conscious chicks in Ca secretion into the intestinal lumen was similar in both groups, but the amount appearing in the gut wall was appreciably greater in CV chicks. 5. There were regional differences in the ability of the gut to absorb Ca. There was a tendency for Ca uptake to be greater in the GF chicks but only in the proximal ileum did this approach significance. 6. When the Ca was confined to an intestinal loop the uptake/g tissue was similar in the proximal ileum of both groups but significantly greater in the distal ileum of the GF group. Uptake/10 mm gut was similar in both groups at both the proximal and the distal size. 7. It is suggested that the absence of a microbial load does not affect the ability of the enterocyte to absorb Ca but the subsequent removal of the absorbed load from the intestinal wall is appreciably slower in the CV group.

A comparison of live and formalised leptospiral microscopic agglutination test.

The sensitivity and reliability of a formalised leptospiral antigen battery was compared with a smaller battery of live leptospiral antigens with 152 human sera positive for leptospirosis. There was good concurrence of results between the formalised and live antigens, however the live battery did fail to detect some positive sera where the antibody level was low. The formalised antigens were more sensitive than the live (P less than 0.001) and more reliable in the detection of early leptospiral infection, but the live antigens were more specific in the determination of probable infecting serogroup.

Isolation of leptospires from a one week dead coypu (Myocastor coypus Molina).

The isolation of L. Interrogans from a one week dead coypu is reported. The isolate was identified as a pathogenic strain of leptospira belonging to the Icterohaemorrhagiae serogroup.

The effects of pregnancy and lactation on the activities of trypsin and alpha-chymotrypsin in the rat pancreas.

1. The food intake, pancreas weight and trypsin (EC 3.4.21.4) and alpha-chymotrypsin (EC 3.4.21.1) activities in the pancreas were measured in rats during pregnancy and lactation and after the young were weaned. 2. All the quantities measured increased significantly during lactation and had returned to their original values by 4 weeks after weaning. Food intake and pancreas weight were highest after the second week of lactation. Total trypsin and alpha-chymotrypsin activity, and the activity per g tissue, fell during pregnancy and rose during lactation, reaching a maximum 1 week after weaning. 3. From these and other results it is suggested that the hypertrophy and hypersecretion of pregnancy and lactation are initiated by changes insulin secretion and mediated by the trophic effects of gut hormones, and that differences in the nature and timing of the response may be controlled by nutrient availability.

Impact of proficiency testing on results of the microscopic agglutination test for diagnosis of leptospirosis.

A proficiency testing scheme for the leptospirosis microscopic agglutination test was provided to 37 laboratories in 23 countries in 2002 (round 1) and to 60 laboratories in 34 countries in 2003 (round 2). Thirty-four laboratories participated in both rounds. Each panel consisted of five rabbit serum samples, four of which were antisera raised against pathogenic serovars of Leptospira. One of these samples was a mixture of two different antisera. The rates of false-negative results, calculated on the basis of the assumption that serovars within a serogroup will cross-react, were 11% for round 1 and 14% for round 2. There were regional differences in the rates of false-negative results. The titers reported by laboratories testing for the same sample with the same serovar varied widely. Laboratories that had previously participated in round 1 reported fewer false-negative results in round 2 than new participants (10 and 21%, respectively [P = 0.002]) and reported 0.56 false-negative results per participant, whereas new participants reported 1.23 false-negative results per participant (P = 0.041). Laboratories that had previously participated also reported fewer false-negative results in round 2 than in round 1 when samples common to both rounds were tested (5 and 15%, respectively [P = 0.028]). The titers reported by the new participants were, on average, lower than those reported by the laboratories that had participated previously (P = 0.019) and were significantly more variable (P = 0.001). Analysis of these results suggests a positive impact of proficiency testing on the testing performance of the participating laboratories.

Experimental infection of monkeys with Leptospira interrogans serovar hardjo.

Grivet monkeys experimentally infected with two different strains of Leptospira interrogans serovar hardjo showed no signs of severe clinical disease. There were no significant macroscopic lesions in any of the tissues examined, but the organisms were demonstrated in various tissues by immunofluorescent technique and were isolated from the blood and urine of two monkeys and the kidney of one. Abraded skin was shown to be a viable route of infection in non-human primates.