Detection of leptospires in urine by PCR for early diagnosis of leptospirosis

Abstract

We tested urine samples from patients at different stages of current leptospirosis and thereafter to determine whether use of the PCR for detection of leptospires in urine can be a valuable alternative to culturing. The procedure of DNA extraction and subsequent PCR applied to 15 freshly voided urine samples proved to be twice as sensitive as culturing. Overall, we were able to detect leptospires in approximately 90% (26 of 29) of the urine samples. Urine and serum samples were obtained from seven patients, before the eighth day of illness. Although it is generally assumed that leptospiruria starts approximately in the second week of illness, we were able to detect leptospires in all of these early urine samples. In contrast, only two of seven corresponding serum samples gave positive PCR results, which suggests that PCR analysis of urine can be more successful for early diagnosis of leptospirosis than PCR analysis of serum. Urine samples from six patients who had been treated with antibiotics at the time of illness were positive by PCR, implying that the patients were still shedding leptospires in their urine despite treatment. Some of these samples were even taken years after the infection, indicating that shedding of leptospires in urine may last much longer than is generally assumed. We conclude that detection of leptospires in urine with PCR is a promising approach for early diagnosis of leptospirosis and may also be useful in studying long-term shedding.

Following the course of human leptospirosis: evidence of a critical threshold for the vital prognosis using a quantitative PCR assay

“…Leptospires are sensitive to a wide range of antibiotics, and the acquisition of new resistances has not been reported. However, standard antibiotic regimens using L-lactamins are not always e¡ective in treating leptospirosis, as a persistent presence of leptospires has been observed in human patients [3,9]. Usual treatments may be unable to remove leptospires from the sites where they are protected from the immune response (meninges, eyes).…”

Classification of Leptospira from the Eyes of Horses Suffering from Recurrent Uveitis

“…Primer pair B64 I/B64 II specifically amplifies a 563-bp DNA fragment of strains from L. kirschneri. PCR conditions, controls, prevention of false-positive reactions because of contamination and Southern blotting to check on the correctness of the products were performed as previously described (Gravekamp et al, 1993;Bal et al, 1994). Prior to PCR, DNA was extracted with the Fast ÔnÕ Easy Genomic Spin DNA purification kit (Anansa, Tebu-bio Laboratories, Cedex, France) according to the manufacturer's recommendation.…”

Development of species-specific PCR primer sets for the detection ofLeptospira

“…To date, various molecular approaches to detect Leptospira have been developed ranging from restriction fragment length polymorphism analysis (Perolat et al, 1993;Kawabata et al, 2001), ribotyping (Perolat et al, 1994), pulsed-field gel electrophoresis (Hermann et al, 1992), DNA hybridization (Zuerner & Bolin, 1997) and variable-number tandem-repeat analysis (Majed et al, 2005) to PCR-based approaches. Identification of Leptospira with PCR has been achieved with arbitrarily primed PCR (AP-PCR) (Perolat et al, 1994;Brown & Levett, 1997;Subarna et al, 2004), low-stringency single specific primer PCR (LSSP-PCR) (Brown & Levett, 1997;Oliveira et al, 2003), PCR restriction endonuclease analysis (PCR-REA) (Brown & Levett, 1997), specific probes (Gravekamp et al, 1993;Bal et al, 1994;Zuerner et al, 1995;Zuerner & Bolin, 1997;Nassi et al, 2003;Merien et al, 2005) and real-time quantitative PCR (Smythe et al, 2002;Levett et al, 2005). Several of the approaches mentioned above allow for the differentiation of species and/or serovars.…”