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PubMed1991

Characterization of serovars of the genus Leptospira by DNA hybridization with hardjobovis and icterohaemorrhagiae recombinant probes with special attention to serogroup sejroe.

Recombinant DNA probes derived from genomic libraries of serovars hardjobovis and icterohaemorrhagiae were applied for the characterization of leptospires. Differences in hybridization signals in combination with the banding pattern appear to provide good characteristics for strain typing. The banding patterns were easy to distinguish, since the recombinant DNA probes hybridized with a limited number of fragments. They were also indicative of genomic relationships between serovars. The probes suggested the existence of four subgroups with extensive genomic homology within the serogroup Sejroe. A number of serovars outside the serogroup Sejroe showed genomic homology with these subgroups. Amplification with the polymerase chain reaction showed a correlation with the genomic homologies demonstrated by Southern analysis. Knowledge about genomic relationships between leptospiral strains, as revealed by Southern analysis, may lead to a more rational approach for primer selection for polymerase chain reaction or cloning of particular genes.

Blotting, Southern↗
PubMed2004

New approach for serological testing for leptospirosis by using detection of leptospira agglutination by flow cytometry light scatter analysis.

Leptospirosis is considered an important reemerging infectious disease worldwide. The standard and most widespread method for the diagnosis of leptospirosis is the microscopic agglutination test (MAT). This test is laborious and time-consuming, and the interpretation of the results is subjective. In the present work we describe an application of flow cytometry (FCM) as a tool for the serological diagnosis of leptospirosis. The analysis is based on the sensitivity of FCM to the size and shape of the bacteria analyzed by measurement of light scatter parameters: forward scatter (FSC) and side scatter (SSC). The addition of positive serum to an infecting leptospiral serovar results in a shift of the light scatter parameter to a different location with higher FSC and SSC values, indicating the formation of leptospiral aggregates. By using immunofluorescent staining, we have shown that the large particles formed are the agglutinated leptospires. Quantification of the agglutination process has been achieved by calculating an agglutination factor (Af), based on changes in the light scatter parameters measured by FCM. Af enables us to determine the specificity of the serological reaction of the patient serum with each leptospiral serovar. In this work, 27 serum samples from 18 leptospirosis patients were tested by both the MAT and the FCM techniques, in which each serum sample was tested against 13 serovars. Twenty-six human serum samples derived from patients with a variety of other defined illnesses were used as negative controls and enabled us to define the Af threshold value as < 9.3 for negative patients, while any value higher than that would be a positive result for leptospirosis. Compared to MAT, the FCM technique was found to be more specific and sensitive, especially in identifying the serogroup in the acute phase of the disease. The whole process was found to be rapid and took less than 1.5 h. Moreover, FCM analysis is objective and can be automated for the handling of large numbers of samples.

Agglutination↗