First isolation of human Leptospira strains, Azores, Portugal.

OBJECTIVES: The aim of this study was the first identification of Leptospira isolates from Azorean inpatients. METHODS: Whole blood samples from 68 inpatients attending the São Miguel Hospital between 2006 and 2008, with a clinical and epidemiological suspicion of leptospirosis, were inoculated in a transport medium broth at the patient's bedside and further processed using a serial dilution technique prior to culture. At admission, 62 (91%) patients were also analyzed for the presence of leptospiral DNA by a nested PCR and 40 (59%) for specific agglutinins by microscopic agglutination test (MAT). The isolates obtained were first assigned at the serogroup level by both MAT reactivity with hyperimmune rabbit antisera and a PCR-based assay with the single primer iRep1. The species identification was performed by DNA sequencing. The use of monoclonal antibodies allowed intraspecific discrimination at the serovar level. RESULTS: Of the 10 (14.7%) human Leptospira isolates, seven were identified as Leptospira interrogans serovar Copenhageni and three as Leptospira borgpetersenii serovar Arborea, which is in agreement with previous data from the Azorean rodent population. CONCLUSIONS: This study represents a great step towards the definitive identification of the pathogenic leptospires in Azorean patients and confirms the bacteriological human-rodent connection for the first time.

Plasmid profile analysis of Portuguese Borrelia lusitaniae strains.

Plasmid profiles of 2 Portuguese Borrelia lusitaniae strains, one isolated from a human patient and the other one from an Ixodes ricinus tick, were obtained by pulsed-field gel electrophoresis to evaluate the plasmid diversity in each strain. Overall, a maximum of 6 plasmids were detected that ranged from 19 kb to 76 kb, revealing completely different plasmid profiles from those previously described for other genospecies of B. burgdorferi sensu lato, the causative agents of Lyme borreliosis. The plasmid location of the ospA gene was investigated by hybridization, allowing its allocation to the plasmid of 70 kb instead of the 54 kb linear plasmid described for B. burgdorferi sensu stricto strains.

A new Borrelia species defined by multilocus sequence analysis of housekeeping genes.

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.

Fine-scale phylogeographic structure of Borrelia lusitaniae revealed by multilocus sequence typing.

Borrelia lusitaniae is an Old World species of the Lyme borreliosis (LB) group of tick-borne spirochetes and prevails mainly in countries around the Mediterranean Basin. Lizards of the family Lacertidae have been identified as reservoir hosts of B. lusitaniae. These reptiles are highly structured geographically, indicating limited migration. In order to examine whether host geographic structure shapes the evolution and epidemiology of B. lusitaniae, we analyzed the phylogeographic population structure of this tick-borne bacterium using a recently developed multilocus sequence typing (MLST) scheme based on chromosomal housekeeping genes. A total of 2,099 questing nymphal and adult Ixodes ricinus ticks were collected in two climatically different regions of Portugal, being approximately 130 km apart. All ticks were screened for spirochetes by direct PCR. Attempts to isolate strains yielded 16 cultures of B. lusitaniae in total. Uncontaminated cultures as well as infected ticks were included in this study. The results using MLST show that the regional B. lusitaniae populations constitute genetically distinct populations. In contrast, no clear phylogeographic signals were detected in sequences of the commonly used molecular markers ospA and ospC. The pronounced population structure of B. lusitaniae over a short geographic distance as captured by MLST of the housekeeping genes suggests that the migration rates of B. lusitaniae are rather low, most likely because the distribution of mediterranean lizard populations is highly parapatric. The study underlines the importance of vertebrate hosts in the geographic spread of tick-borne microparasites.

Human leptospirosis in Portugal: A retrospective study of eighteen years.

INTRODUCTION: Leptospirosis, an under-recognized public health problem, needs to be confirmed through specific laboratory diagnosis. DESIGN: We describe herein a series of 4618 symptomatic patients for whom a microagglutination test (MAT) serology was available, representing a unique picture of human leptospirosis in central mainland Portugal and the Azores islands of São Miguel and Terceira, over eighteen- and twelve-year periods, respectively. RESULTS: The distribution of the 1024 (22%) cases identified was an average 57 cases per year, with higher frequency in males (67%). These represent the majority of leptospirosis notifications in Portugal, with a higher annual incidence rate in the islands, compared to the central mainland (11.1 and 1.7/100,000 population, respectively). Middle-aged adults (25-54 years) were most frequently infected (45%). Cases occurred mainly in December and January. Serovars from nine presumptive serogroups caused infection, with a predominance of Icterohaemorrhagiae, Pomona, and Ballum, accounting for 66% of cases. CONCLUSIONS: Seropositivity was associated with both anicteric and icteric leptospirosis. Several risk factors and a higher transmission risk in certain areas were emphasized. Leptospirosis was confirmed as a steadily increasing public health problem; good surveillance, communication, and laboratory support are thus necessary to reduce the impact of leptospirosis in areas at risk.

Lyme borreliosis in Portugal caused by Borrelia lusitaniae? Clinical report on the first patient with a positive skin isolate.

BACKGROUND: Borrelia lusitaniae was isolated from an Ixodes ricinus tick in Portugal in 1993 for the first time. Further, this borrelia genospecies has been found in ixodid ticks collected around the coasts of southern Portugal and North Africa. Its reservoir has not been defined yet. B. lusitaniae was isolated once until now from a patient with a long standing and expanding skin disorder. PATIENT AND METHODS: A 46-year-old Portuguese woman presented with a skin lesion on the left thigh which had evolved slowly over ten years. The patient reported limb paraesthesias, cramps, chronic headaches, and cardiac rhythm disturbances. History of tick bites was negative nor had the patient ever noticed a skin lesion comparable with erythema chronicum migrans. Skin biopsies were taken for histological evaluation, culture and DNA detection. Antibodies to borrelia were searched by indirect immunofluorescence assay and Western-blot. RESULTS: A bilateral carpal tunnel syndrome and local synovitis was diagnosed. Dermato-histology was normal, serology was negative. Spirochaetal organisms were cultured from a skin biopsy and identified as B. lusitaniae. The patient improved after a 2-week course of intravenous ceftriaxone; the skin lesions did not expand further. CONCLUSIONS: This culture confirmed skin infection by B. lusitaniae in a patient from Portugal suggests an additional human pathogen out of the B. burgdorferi sensu lato complex in Europe, particularly in Portugal.

Lyme borreliosis spirochetes in questing ticks from mainland Portugal.

In Portugal, Ixodes ricinus ticks have been shown to contain DNA of several spirochetes belonging to the Borrelia burgdorferi sensu lato complex, with major differences in the genetic diversity between ecozones. Some isolates have been obtained since 1999, confirming the circulation of pathogenic strains in these ticks. Ixodes ricinus is considered to be a widespread species, however, in Portugal it is found only in a few habitats. Here we present preliminary results from a nationwide survey of questing I. ricinus (n = 4,001) and other Ixodidae (n = 1,534) in Portugal, initiated in 2001. The sampling points (so far 41) were selected using a Geographic Information System, according to the type of vegetation, accessibility and prevalence of human cases. The spatial and temporal of tick abundance and the infection of B. burgdorferi sensu lato in ticks were determined in selected areas. Ticks were examined for the presence of B. burgdorferi sensu lato by culturing (719 out of 4,001 I. ricinus), and direct PCR amplification of the 5S-23S intergenic spacer region (1,870 out of 5,535) followed by RFLP analysis, the reverse line blot assay and nucleotide sequencing of PCR amplicons. The most abundant tick genus was Rhipicephalus (53%), followed by Dermacentor (34%), I. ricinus and Hyalomma (7%, each). The Mafra and Grândola sites, where a more intensive study was carried out, were excellent habitats for I. ricinus. However, a clear difference of the prevalence of Borrelia infection and the genetic diversity of circulating spirochetes was observed in these two sites. Genotyping of all I. ricinus isolates revealed 5 B. garinii, 8 B. lusitaniae and 1 B. valaisiana strains, which were obtained for the first time in these regions along with a considerable percentage of tick-derived PCR amplicons. Two hard-tick species other than I. ricinus in Grândola were also B. lusitaniae positive, thus seeming to take part in the transmission cycle of Borrelia. The seasonal dynamics of I. ricinus in Mafra was bimodal, more pronounced in nymphs than in adults. The present findings indicate that B. burgdorferi sensu lato agents are differentially maintained in nature in local tick populations in different geographic areas across Portugal and that the risk of acquiring Lyme borreliosis in certain areas of Portugal is higher than previously assumed.

The key roles of selection and migration in the ecology of Lyme borreliosis.

The roles of selection and migration of B. burgdorferi s. l. were studied. Questing adult Ixodes ricinus ticks were collected across Europe and analysed for infection with B. burgdorferi s. l. Analysis of the genospecies in individual ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. Segregation of bird- and rodent-associated Borrelia genotypes can be explained by the operation of complement-mediated selection in the midgut of the feeding tick. Phylogenetic analyses of B. burgdorferi s. l. indicate high rates of migration for bird-associated genotypes. Altogether, it is emerging that the ecology of Lyme borreliosis is largely host-driven and that selection and migration are major forces shaping the population structures of B. burgdorferi s. l.