New serovars of Leptospira isolated from patients in Costa Rica: implications for public health.

Leptospira strains JICH 05 and INCIENSA 04 were isolated from hospitalized leptospirosis patients in the province of Puntarenas, Costa Rica. The isolates produced agglutination titres notably against members of serogroups Pyrogenes and Tarassovi, respectively, but appeared serologically unique in the cross agglutinin absorption test (CAAT). Therefore, JICH 05 and INCIENSA 04 were considered to represent two new serovars, designated Corredores and Costa Rica of the serogroups Pyrogenes and Tarassovi, respectively. Multilocus sequence genotyping revealed that both strain INCIENSA 04 and strain JICH 05 belong to Leptospira santarosai. These two new serovars are in addition to various other recently identified highly virulent serovars, including the new L. santarosai, serovar Arenal. Considering the fact that isolation and typing of leptospires from patients has only recently been introduced in Costa Rica, these findings suggest that various known and unknown virulent serovars of Leptospira are circulating in this country and probably beyond, thus posing a severe threat to public and probably veterinary health in the region.

Erratum to: evaluation of the use of selective PCR amplification of LPS biosynthesis genes for molecular type of Leptospira at the Serovar level

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection.

A 285-bp DNA fragment was amplified using the polymerase chain reaction from 38 Leptospira serovars of six different genomic species. The fragments amplified exhibited differential mobilities on nondenaturing polyacrylamide gels resulting from sequence-dependent conformational alterations. Leptospira interrogans serovars could be distinguished from those of other species on this basis.

Evaluation of the polymerase chain reaction for early diagnosis of leptospirosis.

Early diagnosis of leptospirosis is important because severe leptospiral infection can run a fulminant course. The polymerase chain reaction (PCR) was evaluated for the detection of leptospires in clinical samples from patients with acute leptospiral infection. Blood and urine samples from 71 patients with leptospirosis were examined by PCR, culture or serology. Samples from 44 (62%) patients with the diagnosis of leptospirosis were positive by PCR as compared to 34 (48%) by culture. The presence of leptospires was demonstrated by PCR in 13 patients before the development of antibodies, as well as in two patients who were seronegative during their illness and at autopsy. Samples from 16 patients without leptospirosis were seronegative and culture negative, and also negative by PCR. We conclude that PCR is a rapid, sensitive and specific means of diagnosing leptospiral infection, especially during the first few days of the disease.

Simple method for production of internal control DNA for polymerase chain reaction assays for leptospira spp. - abstract

A simple and highly versatile one-step method for the production of internal control DNA for an established polymerase chain reaction assay for Leptospira interrogans is described. The internal control was produced from DNA of serovar bim, and is amplified with the same primers used routinely in our PCR assays. The inclusion of the internal control in the reaction mixture did not affect the efficacy of amplification of the target DNA. The method is simple and rapid and should be adaptable to most PCR assays for Leptospira spp. (AU)

Use of the polymerase chain reaction for rapid diagnosis of leptospirosis - abstract

Leptospirosis is endemic in Barbados with 97 percent of severe cases caused by three serovars of leptospira interrogans. Early diagnosis is important since the disease can run a fulminant course and patients may die before the appearance of characteristic clinical manifestations of Leptospirosis and/or leptospiral antibodies are detected, and therefore the disease may go unrecognized. In this study, the potential of the polymerase chain reaction (PCR) was explored for the early diagnosis of leptospirosis, with a view to detecting leptospirosis within the first ten days of the onset of the disease. Blood and urine samples from 83 patients with leptospirosis admitted to the Queen Elizabeth Hospital, Barbados, between January 1990 and December 1992, were examined serologically, by culture and by PCR. The mortality rate during the study period was 8.4 percent. PCR was more often positive than culture for the detection of leptospires in proven cases by antibody titre and detected the presence of leptospires in sera before the development of antibodies. As culture can take up to 13 weeks, it does not contribute to an early diagnosis. Seroconversion usually occurs on about the seventh day of the disease, thus diagnosis by serology can take a week or more to be decisive. PCR, on the day of admission, and the characterization of PCR products by Southern hybridization can be completed within one or two subsequent days. PCR is potentially a valuable addition to the diagnostic process in leptospirosis (AU)