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.

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 Nino, the main regional climatic phenomenon.

A squirrel monkey model of human T-cell leukemia virus type 1 (HTLV-1) infection was used to evaluate the immunogenicity and protective efficacy of a chimeric peptide vaccine composed of a B-cell epitope from the envelope region (aa 175-218) and three HLA-A*0201-restricted cytotoxic T-lymphocyte epitopes derived from Tax protein (Tri-Tax). These selected Tax peptides induced secretion of gamma interferon (IFN-gamma) in peripheral blood mononuclear cells obtained from monkeys chronically infected with HTLV-1. After immunization, a high titre of antibodies and a high frequency of IFN-gamma-producing cells were detected against the Env and the Tri-Tax immunogens, but not against the individual Tax peptides. This might indicate that epitope(s) distinct from those recognized by humans are recognized by responder monkeys. After challenge, it was shown by competitive PCR that partial protection against HTLV-1 infection could be raised in immunized animals. Further studies should be developed to determine the duration of this protection.

The recent emergence of a decreased susceptibility of Neisseria gonorrhoeae strains to penicillin in New Caledonia has lead clinicians to operate a change in the treatment strategy. In addition, this important health issue has emphasized the need for a rapid means of detecting penicillin resistance in N. gonorrhoeae in order to select an effective treatment and limit the spread of resistant strains. In recent years, the use of fluorescence resonance energy transfer on the LightCycler has proven to be a valuable tool for the screening of mutations occurring in the genome of various microorganisms. In this study, we developed a real-time PCR assay coupled with a fluorometric hybridization probes system to detect a penicillin resistance-associated mutation on the N. gonorrhoeae ponA gene. Following an extensive evaluation involving 136 isolates, melting curve analysis correctly evidenced a 5 degrees C T(m) shift in all N. gonorrhoeae strains possessing this mutation, as determined by conventional sequencing analysis. Moreover, the mutation profiles obtained with the real-time PCR showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Overall, our molecular assay allowed an accurate and reproducible determination of the susceptibility to penicillin corresponding to a mutation present in all chromosomally mediated resistant strains of N. gonorrhoeae.

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.

The susceptibility of Leptospira interrogans serovar icterohaemorrhagiae strain Verdun to selected antibiotics used in medical practice (ampicillin, doxycycline, and ofloxacin) was evaluated in a Syrian hamster model, to determine the efficacy of these antibiotics during the course of the disease. A quantitative PCR assay was used to monitor the density of leptospires in blood and in target organs (liver, kidney, lung, heart, and spleen). Our results demonstrated the ability of ampicillin at a high dose (100 mg/kg of body weight) to clear leptospires from the host, except from kidneys and heart, where 10(2) leptospires/g remained at day 6. Ofloxacin (30 mg/kg) was unable to clear bacteria from blood or kidneys. With doxycycline (10 mg/kg), the clearance of leptospires occurred in 2 days in all the target organs studied, with the exception of liver, which required 3 days. Our data demonstrate the value of monitoring the kinetics of experimental leptospiral infection in order to accurately evaluate the efficacy of antibiotics. We have demonstrated the potential value of doxycycline for the treatment of leptospirosis cases, except in circumstances where it is contraindicated. This experimental model could be used to define better therapeutic strategies for human leptospirosis, by testing associations or new formulations of antibiotics.

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 μl. In a prospective study of human leptospirosis cases, we obtained evidence that a density of 104 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.

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.

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.

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.

BACKGROUNDIn 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.METHODSWe 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.RESULTSA 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.CONCLUSIONThese 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.

ABSTRACT 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.

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.

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.

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.