[Laboratory based human leptospirosis surveillance in New Caledonia (2001-2005)]. / Bilan de cinq années de surveillance biologique de la leptospirose humaine en Nouvelle-Calédonie (2001-2005).

The Pasteur Institute in New Caledonia performs for this territory the biological diagnosis of human leptospirosis; therefore its activity locally gives a rather exhaustive description of this pathology. The results presented here cover the 2001-2005 period and describe the principal epidemiological and biological features of human leptospirosis in New Caledonia. The investigated patients were recruited by the main medical structures: territorial and provincial hospitals, public dispensaries, clinics and general practitioners. The laboratory used the microagglutination test for serological investigations and PCR methods for the early detection of Leptospira genome in clinical samples. 239 cases of leptospirosis were biologically confirmed among 6690 tested patients, giving an average incidence of 21 cases per 100000/year, and a lethality rate of 5.4%. The sex-ratio was 1.8 male/female, patients were predominantly belonging to the 20-50 year age group and were inhabitants from the Northern Province. The circulating serogroups were mainly Icterohaemorrhagiae (69%), Australis (8%) and Pyrogenes (6%). The annual incidence peak occurred in April at the end of the warm season, and the importance of annual outbreaks could be linked with El Ninõ, the main regional climatic phenomenon.

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

In order to follow the course of acute human leptospirosis, an ELISA microtiter plate hybridization method was developed for the quantitative determination of Leptospira spp. in biological samples after PCR. The biotin-labelled amplified product (331 bp from the rrs gene) was hybridized with a complementary capture probe covalently linked onto aminated polystyrene wells, and detected using a colorimetric reaction. The mean detection limit was 50 copies per 10 microl. In a prospective study of human leptospirosis cases, we obtained evidence that a density of 10(4) leptospires per ml of blood is a critical threshold for the vital prognosis of the patients. The practicability of the method makes it suitable for use in tropical areas for multicentric studies. Such studies could lead to a better knowledge of the natural history of the human disease. The method is also suitable for experimental evaluation of improved antibiotic treatments for leptospirosis.

Interest of partial 16S rDNA gene sequences to resolve heterogeneities between Leptospira collections: application to L. meyeri.

This paper describes the advantage of using the first 330 bp (positions 46 to 375, Escherichia coli numbering) of the 16S rDNA gene for comparison of Leptospira isolates. Phylogenetic analysis conducted from the whole 16S rDNA sequences available in databanks as well as that conducted from the partial sequences yielded quite similar results, in accordance with data inferred from previous DNA-DNA relatedness studies. This tool was used for the comparison of Leptospira strains from different reference collections. Consistent results were obtained from the analysis of the polymorphism generated by pulsed-field gel electrophoresis. The study focused on different serovars of L. meyeri species, the classification of which has been controversial. The results revealed large collection heterogeneities, and suggest that the classification of the L. meyeri species should be revised.

Pulmonary haemorrhage as a predominant cause of death in leptospirosis in Seychelles.

We examined the cause of death during a 12-month period (1995/96) in all consecutive patients admitted to hospital with leptospiral infection in Seychelles (Indian Ocean), where the disease is endemic. Leptospirosis was diagnosed by use of the microscopic agglutination test and a specific polymerase chain reaction assay on serum samples. Seventy-five cases were diagnosed and 6 patients died, a case fatality of 8%. All 6 patients died within 9 days of onset of symptoms and within 2 days of admission for 5 of them (5 days for the 6th). On autopsy, diffuse bilateral pulmonary haemorrhage (PH) was found in all fatalities. Renal, cardiac, digestive and cerebral haemorrhages were also found in 5, 3, 3 and 1 case(s), respectively. Incidentally, haemoptysis and lung infiltrate on chest radiographs, which suggest PH, were found in 8 of the 69 non-fatal cases. Dengue and hantavirus infections were ruled out. In conclusion, PH appeared to be a main cause of death in leptospirosis in this population, although haemorrhage in other organs may also have contributed to fatal outcomes. This cause of death contrasts with the findings generally reported in endemic settings.

Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae.

We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.

Factors associated with clinical leptospirosis: a population-based case-control study in the Seychelles (Indian Ocean).

BACKGROUND: In Western countries, leptospirosis is uncommon and mainly occurs in farmers and individuals indulging in water-related activities. In tropical countries, leptospirosis can be up to 1000 times more frequent and risk factors for this often severe disease may differ. METHODS: We conducted a one-year population-based matched case-control study to investigate the frequency and associated factors of leptospirosis in the entire population of Seychelles. RESULTS: A total of 75 patients had definite acute leptospirosis based on microagglutination test (MAT) and polymerase chain reaction (PCR) assay (incidence: 101 per 100,000 per year; 95% confidence interval [CI]: 79-126). Among the controls, MAT was positive in 37% (past infection) and PCR assay in 9% (subclinical infection) of men aged 25-64 with manual occupation. Comparing cases and controls with negative MAT and PCR, leptospirosis was associated positively with walking barefoot around the home, washing in streams, gardening, activities in forests, alcohol consumption, rainfall, wet soil around the home, refuse around the home, rats visible around the home during day time, cats in the home, skin wounds and inversely with indoor occupation. The considered factors accounted for as much as 57% of the variance in predicting the disease. CONCLUSION: These data indicate a high incidence of leptospirosis in Seychelles. This suggests that leptospires are likely to be ubiquitous and that effective leptospirosis control in tropical countries needs a multifactorial approach including major behaviour change by large segments of the general public.

Arbitrarily primed PCR to type Vibrio spp. pathogenic for shrimp.

A molecular typing study on Vibrio strains implicated in shrimp disease outbreaks in New Caledonia and Japan was conducted by using AP-PCR (arbitrarily primed PCR). It allowed rapid identification of isolates at the genospecies level and studies of infraspecific population structures of epidemiological interest. Clusters identified within the species Vibrio penaeicida were related to their area of origin, allowing discrimination between Japanese and New Caledonian isolates, as well as between those from two different bays in New Caledonia separated by only 50 km. Other subclusters of New Caledonian V. penaeicida isolates could be identified, but it was not possible to link those differences to accurate epidemiological features. This contribution of AP-PCR to the study of vibriosis in penaeid shrimps demonstrates its high discriminating power and the relevance of the epidemiological information provided. This approach would contribute to better knowledge of the ecology of Vibrio spp. and their implication in shrimp disease in aquaculture.

Clinical aspects of ocular leptospirosis in New Caledonia (South Pacific).

PURPOSE: The incidence of leptospirosis is very high in New Caledonia (average annual incidence rate: 180/100 000 of the population). To investigate the role of pathogenic leptospires as an aetiological agent of ocular diseases, we report the results of a 5-year survey in New Caledonia. METHODS: We reviewed 13 patients (corresponding to 17 investigated pathologic eyes) retrospectively. The selection of patients was based on epidemiological data, initial clinical examination, biological confirmation of leptospirosis according to reference procedures and a specific polymerase chain reaction assay. The anatomic criteria of uveitis and the criteria describing the evolution of the disease were those recommended by the International Uveitis Study Group. RESULTS: Consequent rates of optic neuritis (65%), posterior uveitis (35%), secluded pupil (24%), interstitial keratitis (18%) and pars planitis (12%) were found. Our rates of recurrence (46%) and of ophthalmic complications (82%) were substantial; some symptoms indicated brain involvement.The final visual damage was severe in 35% of eyes. CONCLUSIONS: Microagglutination and polymerase chain reaction hybridization are complementary tests for the diagnosis of Leptospira-induced ophthalmic lesions. Before deciding on treatment, one must consider the ability of virulent leptospires to persist in immunologically privileged sites such as aqueous humor, cerebrospinal fluid and eukaryotic cells. Polymerase chain reaction is a useful tool for the diagnosis of Leptospira-induced ocular complications, which are probably misdiagnosed if based only on routine laboratory tests. It enables early diagnosis and early specific treatment that now consists of quinolone and cyclines.

In vivo apoptosis of hepatocytes in guinea pigs infected with Leptospira interrogans serovar icterohaemorrhagiae.

To investigate the contribution of the previously demonstrated in vitro apoptosis to the pathogenesis of leptospirosis, guinea pigs were infected with Leptospira interrogans serovar icterohaemorrhagiae strain Verdun and sequentially killed to collect target organs involved in the natural history of the disease (liver, kidneys, lungs, spleen and heart). The combination of histopathological procedures and a specific TUNEL assay showed a significant Leptospira-induced programmed cell death of hepatocytes with a peak at 48 h post inoculation. Hepatocyte nuclei showed morphological changes including fragmented and condensed nuclei. This phenomenon occurred early in the course of the disease at a time where infecting leptospires were present at a low density between the liver parenchyma cells.

Leptospira fainei sp. nov., isolated from pigs in Australia.

Pathogenic leptospires can be causative agents of reproductive problems in pigs. Cultures of uteri and kidneys from two pigs herds in New South Wales and Victoria (Australia) yielded five strains identified as Leptospira on morphological and cultural grounds. Phenotypic characteristics (growth at 13 and 30 degrees C, growth in the presence of 8-azaguanine) were intermediate between those of pathogenic and saprophytic leptospires. No cross-agglutination was observed with reference antisera representing the 24 pathogenic serogroups and the main saprophytic ones. Antiserum against one of the strains did not agglutinate reference stains representative of any serogroup. This provided evidence of a new serovar, designated hurstbridge. Genomic characterization of the five strains was achieved using five molecular approaches. Mapped restriction site polymorphisms in the rrs (16S rRNA) gene were not related to those of any reference strains. Arbitrarily primed PCR fingerprints suggested clonality of the five strains. The strains all showed an identical and unique PFGE profile. PCR, using primers specific for the rrs gene of pathologic leptospires, amplified corresponding sequences from the strains. DNA-DNA hybridization (and reciprocal experiments) using the S1 nucleas/TCA method was performed between one of the strains and the reference strains of Leptospira species. The homology ranged from 0 to 36% (the latter being was Leptospira inadai) thus satisfying the criterion of a new species, Leptospira fainei (type strain BUT 6T). Phylogenetic analysis of 16S rRNA sequence showed that L. fainei and L. inadai formed a clade separate from the previously recognized 'saprophyte' and 'pathogen' clades.

Human leptospirosis in the Seychelles (Indian Ocean): a population-based study.

A leptospirosis surveillance program was carried out for 12 months on the entire population of the Seychelles. Diagnosis was assessed by both microagglutination test and polymerase chain reaction (PCR) assay. In this population of 74,331, leptospirosis was clinically suspected in 125 subjects and confirmed in 75 patients (incidence of 101 per 100,000; 95% confidence interval = 79-126). Leptospirosis was more frequent in middle-aged males with environmental exposure. Eight serogroups were identified and Icterohaemorrhagiae (31%) and Hurstbridge (20%) were the most frequent. Hurstbridge, a recently identified new serogroup, was implicated in severe cases and death. Influenza-like forms accounted for 37% of the cases while jaundice, acute renal failure, and pulmonary hemorrhage occurred in 52%, 28%, and 19%, respectively. Death occurred in six patients and was related to pulmonary hemorrhage. The PCR result was positive after completion of treatment in eight patients, suggesting that the administered five-day course of penicillin may be inadequate to eradicate the bacteria.

Invasion of Vero cells and induction of apoptosis in macrophages by pathogenic Leptospira interrogans are correlated with virulence.

Interactions of virulent Leptospira interrogans serovar icterohaemorrhagiae strain Verdun with Vero cells (African green monkey kidney fibroblasts) and a monocyte-macrophage-like cell line (J774A.1) were assayed by a double-fluorescence immunolabelling method. Infectivity profiles were investigated according to (i) the duration of contact between leptospires and eukaryotic cells and (ii) the number of in vitro passages after primary isolation from lethally infected guinea pigs. Comparative experiments were conducted with the corresponding high-passage avirulent variant and the saprophytic leptospire Leptospira biflexa Patoc I. In Vero cells, virulent leptospires were quickly internalized from 20 min postinfection, whereas avirulent and saprophytic strains remained extracellularly located. In addition, the virulent strain demonstrated an ability to actively invade the monocyte-macrophage-like J774A.1 cells during the early stages of contact and to induce programmed cell death, as shown by the detection of oligonucleosomes in a quantitative sandwich enzyme immunoassay. In both cellular systems, subsequent in vitro subcultures demonstrated a progressive decrease of the invasiveness, pointing out the necessity of using primocultures of Leptospira for virulence studies. Invasiveness of virulent leptospires was significantly inhibited with monodansylcadaverine, indicating that internalization was dependent on receptor-mediated endocytosis. Invasion of epithelial cells and induction of apoptosis in macrophages may be related to the pathogenicity of Leptospira, and both could contribute to its ability to survive in the host and to escape from the immune response.

Public health importance of human leptospirosis in the South Pacific: a five-year study in New Caledonia.

A retrospective study of 192 cases of human leptospirosis in New Caledonia (South Pacific) diagnosed between 1989 and 1993 showed that the disease was endemic throughout the territory. The annual incidence rate was 30 per 100,000 population, and the disease was more frequent in males (67.5%). Cases occurred mainly in March each year. Forty isolates were obtained (20.8%) and identified as belonging to serovars icterohaemorrhagiae (28), pomona (6), pyrogenes (3), ballum (2), and javanica (1). Most cases (54.7%) presented as influenza-like illnesses, while classical Weil's syndrome (fever, jaundice, and renal involvement) occurred in only 15.6% of the patients. Severe ocular complications were found in 3.6% of the patients. Local differences in climate, environment and socioeconomic conditions determined the epidemiologic features. These data emphasize the potential public health importance of leptospirosis in the other insular states in the South Pacific.

Comparison of polymerase chain reaction with microagglutination test and culture for diagnosis of leptospirosis.

Polymerase chain reaction assay (PCR) amplifying a fragment of the Leptospira rrs gene was compared with culture and microagglutination test (MAT) for the diagnosis of leptospirosis in a study of 200 patients with various clinical syndromes compatible with leptospirosis. For the first group of samples tested, PCR identified the 14 cases that later were unequivocally confirmed to be leptospirosis. Thirteen other systemic cases presenting decreasing leptospiral antibody titers were also detected by PCR. The average persistence of leptospiral DNA in serum was estimated at 12 days, with a maximum of 56 days in a culture-confirmed case. The possibility of detecting leptospires in aqueous humor during the ocular complications of the disease was confirmed. The results suggest that PCR is an efficient tool for early diagnosis of leptospirosis during the first 10 days of the disease, especially when the clinical expression of the disease is confusing.

Characterization of Leptospira isolates from serovar hardjo by ribotyping, arbitrarily primed PCR, and mapped restriction site polymorphisms.

Leptospira serovar hardjo isolates of the hardjoprajitno and hardjobovis genotypes were characterized by ribotyping, arbitrarily primed PCR (AP-PCR) fingerprinting, and the study of mapped restriction site polymorphisms (MRSPs) in rrs and rrl genes. After restriction of chromosomal DNA with BglII, EcoRI, or HindIII, each genotype was individualized with a distinct ribotype. The fingerprints produced by AP-PCR with seven primers clearly separated the two groups; primers KF and RSP produced species-specific products which assigned hardjoprajitno and hardjobovis isolates to the species L. interrogans sensu stricto and L. borgpetersenii, respectively. Furthermore, AP-PCR fingerprints gave evidence of a considerable genomic heterogeneity at the strain level among the hardjobovis group. Conversely, the hardjoprajitno group was homogeneous. MRSP profiles in ribosomal genes indicated that hardjoprajitno and hardjobovis isolates belonged to L. interrogans MRSP group B and L. borgpetersenii group C, respectively. AP-PCR and determination of MRSPs in ribosomal genes proved to be quick and reliable methods for typing Leptospira strains and for studying intraspecific population structures.

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.