Distribution of Streptococcus pneumoniae serotypes responsible for penicillin resistance and the potential role of new conjugate vaccines in New Caledonia.

Invasive pneumococcal disease is a significant cause of morbidity and mortality worldwide. The aim of this study was to establish the serotypes responsible for pneumococcal disease and the serotypes responsible for penicillin resistance in Noumea, New Caledonia. Isolates of Streptococcus pneumoniae from all body sites referred to the Microbiology Department of the Pasteur Institute in New Caledonia between May 1999 and May 2001 had serotyping and susceptibility testing performed. Basic demographic data on patients were also collected. A total of 298 isolates were included in the study. The most common serotypes were types 1 (20%), 23F (10%), 12F (8%), 19F (8%), and 6B (5%). The serotype distribution differed significantly with age, site of collection, and ethnicity. Overall, 280 of 298 (94%) of the isolates had serogroups that are included in the 23-valent vaccine. However, only 14 of 20 (70%) of the isolates associated with invasive disease from children younger than 5 years of age and 26 of 113 (23%) of invasive isolates from patients more than 5 years of age were included in the new 7-valent conjugate vaccine. Overall, reduced susceptibility to penicillin was found in 43 of 298 (14.4%) of the isolates, with 13% falling into the intermediate resistance category and only 5 (2%) being high-level resistant. A higher prevalence of penicillin resistance occurred in younger age groups and in European patients and involved specifically certain serotypes. The 7-valent conjugate pneumococcal vaccine has potential benefit for the New Caledonian population under 5 years of age and should be considered for future vaccines schedules.

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.

[Detection of leptospirosis reservoirs in Madagascar using the polymerase chain reaction technique]. / Recherche de réservoirs de la leptospirose à Madagascar par la technique d'amplification génique.

A polymerase chain reaction (PCR) technique was used for detection of the Leptospira interrogans rrs gene in kidney tissue from 115 rats, 50 zebu cattles and 13 pigs in an attempt to identify a possible animal reservoir of leptospirosis in Madagascar. In addition, serological testing of 105 individuals in close contact with animals was carried out. The PCR analysis was negative for all the samples tested and only one person was found seropositive at a low titer. The findings suggest that leptospirosis, if prevalent in Madagascar, is likely rare.

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.

Field evaluation of a one-step dipstick assay for the diagnosis of human leptospirosis in the Seychelles.

OBJECTIVE AND METHOD: To compare the response of a dipstick assay (DSA) detecting Leptospira-specific immunoglobulin M (IgM) antibodies with that of an enzyme-linked immunosorbent assay (ELISA), an indirect haemagglutination assay (IHA), the microagglutination test (MAT) and a polymerase chain reaction assay (PCR) in patients with leptospirosis confirmed by MAT alone or by MAT and/or PCR (MAT/PCR). RESULT: In 75 patients with acute leptospirosis diagnosed by MAT (respectively, 90 patients diagnosed by MAT/PCR), the response in paired early and convalescent sera was positive in 78.9% (67.9%) by DSA, 76.0% (67.8%) by ELISA, 58.7% (55.6%) by IHA, 44.0% (53.3%) by PCR, and 100% (90.0%) by MAT. In early serum only, the response in patients diagnosed by MAT (respectively by MAT/PCR) was positive in 36.0% (38.9%) by DSA, 36.0% (37.8%) by ELISA, 14.7% (18.9%) by IHA, 39.2% (48.3%) by PCR, and 53.3% (58.9%) by MAT titre > or =1:100. DSA detected the main serogroups implicated in human leptospirosis in Seychelles and demonstrated sensitivity comparable to ELISA. In 124 single sera from control subjects without overt disease, the response was positive in 4.8% by DSA, 3.2% by ELISA, 3.2% by IHA, 13.8% by PCR, 37.9% by MAT titre > or =1:100, and 2.4% by MAT titre > or =1:800, giving evidence of the frequency of both past and current subclinical infection in Seychelles and that DSA was less sensitive than MAT to detect moderate levels of leptospiral antibodies. CONCLUSION: DSA is a simple and reproducible assay well adapted to field conditions and could usefully contribute to the evaluation of leptospirosis in areas devoid of serological laboratory facilities.

[Epidemiologic and clinical study of leptospirosis in Bourail (New Caledonia)]. / Etude épidémiologique et clinique de la leptospirose à Bourail (Nouvelle Calédonie).

Leptospirosis is a frequent zoonosis in New Caledonia, mostly in the Bourail area, with an incidence of 9.5/1000 inhabitants. This town is an important cross-roads between the main town, Noumea, and the bush. An agricultural show takes place every year in this town, concentrating a great number of domestic animals and several thousand people coming from all over the island. A study was carried out on 78 patients with leptospirosis. The patients were mostly men (70%) of all ages of Melanesian and European origin. People who were living in the bush were more often attacked by leptospirosis than people living in towns. There were two annual peak values (March-April and July-August). Three symptoms (fever, headache, myalgia) were frequent (56%). Leucocytosis increased in 45% of cases. The serogroup icterohaemorrhagiae was the more common (38%). After treatment by penicillin, patients recovered in 83% of cases, but a fatal outcome occurred in 4 patients. An information campaign was necessary in order that people seek medical care as soon as the first symptoms appeared. At present, vaccination does not seem to be possible in such a context.

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.

Serological titres to Leptospira fainei serovar hurstbridge in human sera in Australia.

A set of 723 diagnostic sera from human patients, submitted for the microscopic agglutination test (MAT) for antibodies to a group of 6 leptospiral serovars, was also tested by MAT for antibodies to the recently-discovered Leptospira fainei serovar hurstbridge. MAT titres of > or = 128 to serovar hurstbridge were detected in 13.4% of these sera, and titres of > or = 512 in 7.2%. In contrast, none of 62 sera obtained from a control population of laboratory staff gave titres of > or = 128. The difference between the number of titres of > or = 128 given by the two groups of sera was highly significant (P < 0.01). The titres observed may have been due to cross-reactions with other leptospiral serovars, but this could not be demonstrated. An alternative explanation is that serovar hurstbridge is present in the human population.

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.

Human leptospirosis in the Mekong delta, Viet Nam.

To estimate the seroprevalence of human leptospirosis in the Mekong delta in Viet Nam, an epidemiological survey was conducted in the province of Tien Giang, which is representative of the socioeconomic activities of the region (rice growing and cattle breeding). A cross-sectional study included 35 clusters representing 1400 people randomly selected and aged 15-60 years. Sex, age, occupation, contact with animals, type of water supply, and individual habits were recorded. Leptospiral agglutinins were detected by the microagglutination test, with a battery of 22 live antigens representing the main pathogenic serogroups of Leptospira species and additional local strains. 263 sera (18.8%) gave positive results and 41 (2.9%) had a titre of agglutinins > or = 400, suggesting recent infection. No significant difference was found between females and males. The distribution of seroprevalence was homogeneous throughout the population studied, with the exception of the 15-25 years age group, in which leptospiral antibodies were less frequent. Fifteen serogroups were found, the most prevalent being Bataviae (21.7%), Panama (15.2%), Icterohaemorrhagiae (13.7%) and Australis (8.7%). No significant link between leptospiral seropositivity and professional activities or contacts with animals was found, indicating that leptospirosis in the Mekong delta may be considered as an environmentally linked disease. Leptospirosis is known to be endemic in south-east Asia, and these data demonstrated the high level of circulation of leptospires and the potential importance of leptospiral infections among the rural population in this area.

Epidemiology of leptospirosis in New Caledonia (South Pacific): a one-year survey.

We describe a series of 144 cases of leptospirosis diagnosed in 1989 in New Caledonia. The incidence rate was 90 per 100,000 person-years, with a specific mortality rate of 4% patients. Those affected (100 males, 44 females) were mainly aged 20 to 40 years. Incidence in rural areas (112 per 100,000 person-years) was seven times higher than in urban settlements. Two periods with higher incidence were noticed corresponding to highest rainfall. Twenty-nine of the cases occurred in individuals with professions commonly associated with leptospirosis. Contacts with rats, dogs and ditch or river water were the most frequently mentioned. The clinical expression of the disease was polymorphic: 60% of the patients had mild symptoms, 40% were acute forms including Weil's disease. Of 57 hospitalized, 23% were admitted with an initial diagnosis of dengue, and 37% with leptospirosis. Main clinical syndromes were: icterus and/or renal syndrome in 50% of patients, cardiac syndrome in 65%, acute myalgies in 58% and pulmonary syndrome in 50%. Although hemorrhages were uncommon (17%), 40% of the cases demonstrated thrombocytopenia (< 50,000/m3). Pancreatic involvement with hyperamylasemia was evidenced in 50% of cases. Twelve serogroups of Leptospira were implicated, Icterohaemorragiae predominated (41%), but was not associated with severe forms. In New Caledonia, like in all tropics, leptospirosis must be considered as an environmental diseases, professional activities being just an additional risk factor. Use of serology as a reliable tool for confirmation of cases in areas of high environmental contamination is discussed.

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.

Rapid identification of pathogenic Leptospira species (Leptospira interrogans, L. borgpetersenii, and L. kirschneri) with species-specific DNA probes produced by arbitrarily primed PCR.

Arbitrarily primed PCR (AP-PCR) assays can be used to discriminate between species of Leptospira. Comparative analysis of the fingerprints obtained from representative sets of serovar reference strains of Leptospira interrogans sensu stricto, L. borgpetersenii, and L. kirschneri and the reference strains of the other Leptospira spp. revealed species-specific DNA fragments. These species-specific sequences were reamplified in order to produce digoxigenin-11-dUTP-labeled genomic DNA probes that could be used to identify Leptospira species. Three probes (specific for L. interrogans sensu stricto, L. borgpetersenii, and L. kirschneri) were selected and tested with 72 representative serovar reference strains, all of which had previously been studied by DNA-DNA hybridization. The two techniques were in general agreement, and hybridization with AP-PCR-derived probes was shown to be a useful approach for rapid species determination of leptospires, without the prior need for DNA sequence information. These nonradioactive probes can be used to identify Leptospira species in nonspecialized laboratories, and this should contribute to a better knowledge of the molecular epidemiology of leptospirosis.

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.