Severe leptospirosis in hospitalized patients, Guadeloupe.

We evaluated prognostic factors for leptospirosis in 168 consecutive hospitalized patients in Guadeloupe. Factors independently associated with severity included chronic hypertension or chronic alcoholism, late initiation of antibacterial therapy, abnormal chest auscultation results, icterus, oligoanuria, disorders of consciousness, elevated aspartate aminotransferase levels, hyperamylasemia, and Leptospira interrogans serovar Icterohemorrhagiae.

Borrelia burgdorferi sensu lato diversity and its influence on pathogenicity in humans.

Among the Spirochaetes, the Borrelia burgdorferi sensu lato complex is responsible for Lyme borreliosis. This complex comprises more than 13 Borrelia species. Four of them are clearly pathogenic for humans: B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. spielmanii. They can generate erythema migrans, an initial skin lesion, and can then spread deeply into the host to invade distant tissues, especially the nervous system, the joints or the skin. In humans, Borrelia pathogenicityseems to be linked with taxonomic position, but in vitro studies show the role of plasmids in B. burgdorferi s.l. pathogenesis. The inter- and intraspecies genetic diversity of B. burgdorferi s.l. evidences a clonal evolution of the chromosome, while plasmid genes are quite variable, suggesting their major role in Borrelia adaptability. The plasmid-encoded adhesins and vlse, crasps and osp genes determine invasiveness and host immune evasion of B. burgdorferi s.l., and select the bacterial host spectrum. The geographic distribution of B. burgdorferi s.l. is closely related to its vectors and competent hosts, and its development within these influences its diversity, taxonomy and pathogenesis, primarily via genetic lateral transfer.

Multilocus sequence analysis of atypical Borrelia burgdorferi sensu lato isolates--description of Borrelia californiensis sp. nov., and genomospecies 1 and 2.

Taxonomy of Borrelia burgdorferi sensu lato (s.l.) was recently improved by the use of multilocus sequence analysis (MLSA), a new approach to replace the cumbersome DNA-DNA hybridization method [Richter et al., 2006. Int. J. Syst. Evol. Microbiol. 156, 873-881]. In this study, we used this methodology to classify B. burgdorferi s.l. strains isolated both in Europe and the United States, the exact taxonomic status of which remained unclear. We conclude that MLSA can surpass the discrimination power of whole DNA-DNA hybridization, and we delineate three new North American B. burgdorferi s.l. species. In contrast, European atypical strains constituted a subgroup of B. burgdorferi sensu stricto (s.s.).

Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis.

Leptospirosis is a worldwide-distributed zoonosis, endemic in tropical areas. Epidemiologic investigations of leptospirosis still rely on tedious serological identification tests. Recently, molecular typing systems based on variable-number tandem-repeat (VNTR) analysis have been described and have been used to identify Leptospira interrogans strains. Although L. interrogans is the most common Leptospira species encountered in human infections around the world, other pathogenic species, such as Leptospira kirschneri and Leptospira borgpetersenii, are also frequently associated with human leptospirosis. In this study, we aimed to extend multilocus VNTR analysis (MLVA) identification of strains to species other than L. interrogans. We designed primers for VNTR loci found in L. interrogans, L. kirschneri, and L. borgpetersenii. The discriminatory power of the redefined primers was evaluated on collection strains and then on clinical strains. We also carried out a retrospective study on 156 strains isolated from patients and animals from New Caledonia, an area of high endemicity in the South Pacific. Our results show that this simple PCR-based MLVA typing technique is a powerful methodology for the epidemiology of leptospirosis.

Partial rpoB gene sequencing for identification of Leptospira species.

The usual target for sequence-based identification of Leptospira species is the 16S rRNA gene. However, because the 16S rRNA gene is not polymorphic enough, it is necessary to sequence a 1500 bp segment of this gene for accurate identification. Based on the alignment of previously determined rpoB of three Leptospira strains, we designed and tested a primer pair that enabled us to amplify and sequence a 600 bp segment of Leptospira rpoB. This segment was species-specific for the 16 species tested, but was unable to separate Leptospira interrogans serovars accurately. For the 11 L. interrogans serovars tested, only seven genotypes could be determined. We thus think that analysis of partial rpoB may be useful as an initial screening test for the identification of a new isolate of Leptospira and detection or identification of Leptospira in clinical or environmental samples, but not for serovar determination.

A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence.

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.

Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov.

To evaluate multilocus sequence analysis (MLSA) for taxonomic purposes in the delineation of species within Borrelia burgdorferi sensu lato, seven relevant loci of various strains for which extensive DNA-DNA reassociation data were available were sequenced. MLSA delineation proved to be fully concordant with conventional methods. Our analysis confirmed the delineation of a novel species, Borrelia spielmanii sp. nov., previously known as 'Borrelia spielmani' Richter et al. 2004, with strain PC-Eq17N5T (=DSM 16813T = CIP 108855T) as the type strain.

Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France.

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m(2), respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m(2)), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.

Short report: dual infections with Puumala virus and Leptospira interrogans serovar lora in a bank vole (Clethrionomys glareolus).

Leptospirosis and hemorrhagic fever with renal syndrome are public health problems in Croatia. Diagnosis and epidemiology of these diseases are complicated because these two diseases are sympatric in certain areas. We describe a natural dual infection of Puumala virus and a leptospire in a bank vole (Clethrionomys glareolus).

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity.

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.

Trends in leptospirosis epidemiology in France. Sixty-six years of passive serological surveillance from 1920 to 2003.

OBJECTIVES: The incidence of leptospirosis, a worldwide zoonosis, has significantly decreased in Western Europe during the last decades. Our aim was to analyse all reliable data collected at the Institut Pasteur from 1920 to 2003 to evaluate more precisely the evolution of the incidence of leptospirosis in France. METHODS: The passive surveillance system used as early as 1920 allows the evaluation of leptospirosis incidence. Serological results inferred from the microagglutination procedure were used to evaluate variation in the incidence of the disease and the evolution of the Leptospira serogroups involved in the human disease. RESULTS: No significant variation either in the number of leptospirosis cases or in the incidence of the disease was observed. However, the period of the 1970s was characterized by a rather low incidence. The weather plays a major role by modifying fresh water abundance, rodent populations and human behaviour. However the weather's influence is not the sole factor involved in the incidence rate. No cyclic variation was evident. CONCLUSION: Although France has the highest incidence of leptospirosis in Europe, the analysis of serological data collected over 66 years has allowed us to conclude that in France, the incidence is slowly decreasing.

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis.

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.

Characterization of Borrelia lusitaniae isolates collected in Tunisia and Morocco.

Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato and is infrequently isolated in Europe. In contrast, this species is by far the most predominant in North Africa and in Portugal. In this study, we analyzed the genetic diversity, at several loci, of a large population of isolates from free-living Ixodes ricinus ticks collected in Tunisia and Morocco. We found a moderate diversity of the whole genome by using pulsed-field gel electrophoresis as well as in the ospA gene sequences, compared to a high level of strain homogeneity in the small noncoding ribosomal spacer. In contrast, a high diversity of this locus has been previously reported for Portuguese isolates. We hypothesize that B. lusitaniae strains isolated in North Africa constitute a clone of Portuguese origin.

Identifying relapsing fever Borrelia, Senegal.

We describe a nested polymerase chain reaction for the identification of Borrelia species from serum of patients with unidentified fevers. This technique, based on single nucleotide polymorphisms of the 16S ribosomal RNA gene, was used to test blood samples from 7,750 patients, 33 of whom were diagnosed with spirochete infections. Borrelia crocidurae was the only species identified.

Detection and identification of Ehrlichia spp. in ticks collected in Tunisia and Morocco.

A broad-range 16S rRNA gene PCR assay followed by partial sequencing of the 16S rRNA gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa.

Risk factors for leptospirosis in metropolitan France: results of a national case-control study, 1999-2000.

Risk factors for leptospirosis in France were investigated to improve the vaccination program for this disease. Data from 90 hospitalized case patients and 169 matched control subjects were analyzed in a case-control study. Skin lesions, canoeing, contact with wild rodents, and country residence were independently associated with leptospirosis, emphasizing that leisure activity is a risk factor for this illness.

Genetic diversity among Borrelia strains determined by single-strand conformation polymorphism analysis of the ospC gene and its association with invasiveness.

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three Borrelia species: Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac.). Previous research based on analysis of ospC sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted ospC groups by testing human clinical strains isolated from disseminated forms. To screen for ospC genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known ospC sequences from 44 different strains were first tested, revealing that each ospC group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose ospC sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of B. afzelii, seven of B. garinii, and one of B. burgdorferi sensu stricto. This study confirmed that invasive strains are not distributed among all 69 ospC groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of B. burgdorferi.

Molecular diversity of the ospC gene in Borrelia. Impact on phylogeny, epidemiology and pathology.

Lyme borreliosis is a 2 steps disease: i) Localized erythema migrans ii) occasionally a disseminated disease. Three out of the 10 up to now described Borrelia species are pathogenic for man and each of them exhibits its own organotropism: joints for Borrelia burgdorferi sensu stricto (B.b. ss), nervous system for B. garinii, skin for B. afzelii, ospc gene is subject to lateral transfer leading to a huge diversity among corresponding encoded proteins. This allows the spirochete to develop a repertoire of epitopes to escape the host immune response. We noticed that the European endemic ospc repertoire is only a subset of the American one. This bottleneck situation transduces a "founder's event" suggesting B.b. ss has been imported from North America to Europe at historical times. Another valuable observation is the fact that isolates from disseminated forms (called "invasive") of the disease, all are distributed in only ten out of the 70 ospc genotypes. The conclusion is that in human, some OspC conformations are associated with the invasive potential of a given Borrelia isolate.

Strain typing of Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii by using multiple-locus variable-number tandem repeat analysis.

Human Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by Borrelia spirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of Borrelia burgdorferi and subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse Borrelia isolates including B. burgdorferi, B. afzelii, and B. garinii. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (D) values ranging from 0.10 to 0.87. The average D value was 0.53 across all VNTR loci. A clear correlation exists between the repeat copy number and the D value (r = 0.62) or the number of alleles (r = 0.93) observed across diverse strains. Cluster analysis by the unweighted pair-group method with arithmetic means resolved the 30 observed unique Borrelia genotypes into five distinct groups. B. burgdorferi, B. afzelii, and B. garinii clustered into distinct affiliations, consistent with current 16S rRNA phylogeny studies. Genetic similarity and diversity suggest that B. afzelii and B. garinii are close relatives and were perhaps recently derived from B. burgdorferi. MLVA provides both phylogenetic relationships and additional resolution to discriminate among strains of Borrelia species. This new level of strain identification and discrimination will allow more detailed epidemiological and phylogenetic analysis in future studies.