Leptospira

Leptospirosis are zoonoses, transmissible to humans, caused by bacteria of the genus Leptospira. The disease was described by Weil as an infectious relapsing jaundice. The pathogen was discovered by Inada in Japan in 1915.

I – CLASSIFICATION:

Belonging to the order of Spirochetales the genusLeptospira alone constitutes the family LeptospiraceaeThere are two species. L.interrogans, pathogen, andL.biflexa, saprophyte and aquaculture, found in stagnant water. There is no homology for DNA of the two species.

Leptospira are divided into more than 200 serovars that differ only in their antigenic properties. Depending antigenic communities serovars are grouped into serogroups (23).

Serogroups most often encountered in France are:

Icterohaemorrhagiae and Grippotyphosa, Canicola, Australis, Pomona, Ballum, Autumnalis and Bataviae.

II – HABITAT AND EPIDEMIOLOGY:

Leptospira are widespread in nature. They have a worldwide distribution.

The reservoir consists of wild animals, especially rodents, which are healthy carriers of leptospires in the kidneys and eliminate these bacteria in their urine.

In the external environment, leptospires can survive and multiply if conditions are favorable, stagnant water, slightly alkaline pH, presence of organic compounds, sludge, vases. These conditions are realized in ponds, swamps, sewers, rice fields, mines.

Contamination. Humans can become infected through direct contact with animals (rats, pigs, horses, cattle).Leptospirosis is an occupational disease of sewer workers, farmers, slaughterhouse workers, butchers, minors.Indirect contamination is the most common (60% of cases). It is done by penetration of the germ in skin softened by a prolonged stay in the water. This is often a leisure sickness on the edge of freshwater fishing, swimming, camping.

Mucocutaneous contamination (conjunctival or throat) or digestive possible.

In France, there is a very strong seasonal peak in July and August.

The number of reported cases is probably less than the actual incidence of this disease.

Furthermore, in many cases, it is likely that the diagnosis of leptospirosis is not worn.

The relative frequency of leptospirosis in Reunion to report.

III – CHARACTER BACTERIOLOGICAL:

A – Morphology and mobility:

– It is a flexible micro-organism, helical, thin, cross-division

– Along a few microns to 40 microns (usually 6-20 pm) on 0.1 | Jm width,

– The ends may be curved or hook (biflexa or interrogans)

– Alternative Mobility rotation, flexion, translation.

1. Light micrographs:

These bacteria are not visible in the bright field microscope, but are visible under the microscope the black background or phase contrast microscope, or by special stains:

– Fontana silver impregnation-Tribondeau,

– Giemsa Vago.

2. Examination by electron microscopy:

We recognize different structures:

– A protoplasmic cylinder limited by a carbohydrate-peptide membrane (Figure 1)

– Two independent axial filaments similar to flagella, each inserted from one end to the opposite parities subterminal protoplasmic cylinder.

Their structure is identical to flagella of Gram-negative bacteria,

– An outer membrane enveloping the whole body,

– Nuclear material, ribosomes and lamellar structures resembling mesosomes are recognized.

B – Vitality:

It is important despite the relative fragility of the germ.

Prolonged survival – in the soil, the water at an alkaline pH (7.7),

– Cold,

– Freeze-drying,

– The temperature of the laboratory environment Reiter and Rame with 20% rabbit serum coated with petroleum jelly.

Survival is 2 to 3 days in the sewage, 18-20 hours in seawater.

C – cropping characters:

All leptospires can be cultured in serum-containing medium. In general, they grow at 30 ° C in the presence of oxygen. They are aerobic, but the culture is promoted by a slightly enriched in CO ^ atmosphere.

Nutrient requirements: the long chain fatty acids (24 carbons or more) are needed as a carbon and energy source.The ammonium rather than amino acids brings nitrogen. Purine, thiamine (vit. B 1) and cyanocobalamin (vit. B12) are necessary. The Ca ++ and Mg ++ promote growth.

A temperature of 29 ° C and a slightly alkaline pH (7.2-7.6) are the optimum conditions.

D – Substances produced:

– For some: toxin. A carbohydrate-lipid-polypeptide moiety to endotoxin nature was reported (Patoc 1)

– Certain serovars produce hemolysin such panama, and grippotyphosa australis,

– Enzymes: esterase, oxidase, urease, transaminase, amidase …

E – Antigens:

II exist three antigens:

– H antigen, protein “flagellar” of axial filament has a role in the reaction of agglutination-lysis

– Antigen 0, polysaccharide, of the “somatic” wall which has a role in immunity by inducing antibodies leptospiricide and protective effect,

– Surface antigen of an unknown nature, located in the casing and has a role in the agglutination-lysis.

Strains in the presence of heterologous antiserum undergo antigenic variation.

The pathogenic species L. interrogans serogroups are 20 including 16 recognized by the WHO, serogroups:

– Icterohaemorrhagia ;,

– Canicola,

– Autumnalis,

– Hebdomadis, Bataviae, Panama …

Serogroups and serovars are shown in Table I.

In the species L. biflexa, we recognize two serogroups.

IV – PATHOGENICITY:

A – pathogenic natural power:

The haemorrhagic icterus leptospirosis or Weil’s disease and Mathieu is not the most common form. Jaundice missing in 80% of cases of leptospirosis and hepato-nephritis exists only in the most severe cases, usually due to L.icterohoemorrhagioe.

The febrile syndrome suddenly begins 4-12 days after infection (see diagram). It corresponds to a septicemic stage lasts 5-7 days. After clinical improvement, shorter febrile relapse occurs about the 15th day. Defervescence is observed towards the 20-25 ^th day (Figure 2).

Other suggestive signs are a pain syndrome (myalgia), meningeal irritation and conjunctival injection.

Mortality in France is about 10% of cases. It is higher overseas territories.

The clinical forms of the main serogroups in France are listed in Table II. Polymorphism is clinically important.

B – Experimental pathogenicity:

The young guinea pig is the animal of choice. Inoculated intraperitoneally with blood or urine of a patient, the animal becomes feverish in three or four days. Then appear jaundice and bleeding. The death of the animal occurs in less than two weeks. Autopsy (dangerous for the handler) retrieves leptospires in abundance in the liver, kidneys, blood and urine.

V – BIOLOGICAL DIAGNOSIS OF LEPTOSPIROSIS:

A – Direct diagnosis:

The interest of the various examinations that highlight leptospires is presented in the table below.

In fulminant where death occurs in the early days, the diagnosis can only be made by isolation of the germ. Gene amplification in vitro detection in pathological material is under development.

1. Search by crops:

In vitro: most pathogenic strains can be isolated without too much difficulty provided you use suitable samples, performed before antibiotic therapy, and ridding the pathological product from other contaminating microorganisms (dilution, 5-fluorouracil, filtration …).

Strict working conditions must be met:

-verrerie no trace of detergents or antiseptic and washed as for virology (cell culture). Serum used must be checked for absence of antibodies antileptospires etc.

– The incubation temperature is 28-30 ° C.

– The pH 7.2-7.6.

– The selection of a culture medium; may be used in different types of environments:

a / Liquid media for passage and maintenance of stem

– Reiter and Ramme added rabbit serum

– Vervoot, Korthof,

– Stuan more recently marketed (asparagine, glycerin, 10% rabbit serum).

b / agar media

– Solid medium Noguchi, abandoned,

– Chang medium semi-solid (bactotryptose, liver extract, horse serum and hemoglobin)

– Middle of Cox and Larson interesting

c / serum-free media

– Mid Babudieri,

– Mid Mailloux of Shenberg chemically defined, hence the interest in these environments for metabolic study, preparation of antigens and vaccines.

crops should be monitored very closely by microscopic research of leptospires in the 6th, 15th, 21st, 30th day, and then every 15 days for two months by the following techniques: dark-field microscopy, fluorescence technique, silver staining.

Once grown, leptospires must be identified and classified primarily in cash:

L. interrogans: pathogens, L. biflexa: saprophytes, with simple biochemical reactions.

The isolated strain is examined in agglutination-lysis:

– With group antisera,

– Then with the antisera serovars.

2. Animal inoculation:

This is a crucial time because it is the direct method more sensitive but only some serovars determine a deadly disease in the guinea pig (L. icterohoemorrhagioe, L. autumnalis), in other cases there is only a leptospirémie.

In addition to the guinea pig, hamster can be useful especially for L. canicola.

Animals must be selected youth and low weight.

The inoculation is done by intra-peritoneal route of 0.5 to 1 ml of pathological product.

The animals were monitored clinically, weighed, and the temperature taken two times per day. Examination of peritoneal fluid is recommended.

Post mortem (after spontaneous death or sacrifice to day 21), we practice without delay the exam after autopsy. The tissue is cultured (liver, kidney, spleen) and practical microscopic examination. On serum serology can be practiced.

B – Serological diagnosis:

The achievement by leptospira gives a solid and lasting immunity. From the 8th day, antibodies are usually found, but their appearance may be delayed if the patient received early antibiotic therapy.

The indirect diagnosis has two technical groups.

1. screening reactions which are:

a / The Complement fixation reaction:

Group reaction regardless of the antigen used. It requires a good antigen makes leptospires merthiolatés and stabilized suspension.

b / The macroscopic reactions:

– Either capillary tubes (technique Stoenner)

– On board with:

– Formulated antigens mixed (technical Galton)

– TR or heat-antigen antigens (Read 4 minutes).

c / The hemagglutination reaction:

Made microplate, it is a quantitative reaction using sheep red blood cells sensitized by a soluble antigen (HA). The antigen HA is an alcoholic extract of Leptospira biflexa. The hemagglutination reaction is quantitative and is tested by microtiter.

d / microscopic reaction using antigen biflexa Patoc (leptospiral aquaculture):

It requires ongoing maintenance of live strain and a good dark-field microscopy. Serovar L. biflexa often cross-reacts with sera from patients with leptospirosis.

All these detection reactions can be positive from the 8th day of the disease, but they négativent earlier than the agglutination-lysis technique.

2. Reference reactions (confirmation):

This is the agglutination and lysis reaction (ADR) with reading in dark field microscope practiced with a wide range of antigens:

– Either the classic serodiagnosis of Martin and Pettit,

– Either the CDC variant that requires special microscopic equipment.

The reaction is to bring together the cultures of various leptospira serovars (antigens) and the patient’s serum dilutions (1 / 10®, 17100th, 000th 1/1) with a control culture without serum tube. After 2 hours of oven, one drop of each tube is examined in black. If the response is positive, the presence of antibodies leads to an 00 1/1 by clumping (agglutination) and 1 / 1000par of these clusters devices erosions (lysis).

This reaction known errors:

– Default: too early serum unusual serotype, early treatment with antibiotics or corticosteroids,

– By excess: because of the persistence of antibodies old, due to coagglutinations.

3. Other reactions:

Tests using ELISA were conducted in order to overcome the two factors limiting the RAL namely the use of live strains and darkfield microscope.

ELISA is less sensitive than the RAL, although seroconversion ELISA IgG-RAL is quite parallel. The IgM ELISA method would allow earlier detection. The Western blot yielded results with L. icterohaemorrhagiae protmetteurs.

VI – TREATMENT:

A – Prophylaxis:

This is a global problem.

– Preventive measures include both the fight against rodents and contaminated animals, sterilization of stagnant water, monitoring of water bodies, protection of exposed subjects (boots, gloves …), serological surveys on farms to eliminate carriers.

– Leptospiroses are occupational diseases recognized as such.

– The vaccinating preparations must meet specific sérotyvars to the area and be versatile. In France the human vaccination is recommended for professional presentations. A vaccine using an attenuated strain is studied.

B – Curative:

Leptospira are sensitive to penicillin, chloramphenicol, tetracycline and streptomycin. Macrolides are also effective.

The treatment should be extended 2-3 days after return to normal temperature.

The effectiveness of treatment depends on its early; its effectiveness is questionable when the lesions are hépatonéphrites.

The treatment is justified only for severe leptospirosis (ictérohémorragiques, the Far East, Japanese and L. canicola).

Many benign leptospirosis do not need treatment. Adjuvant treatments are undertaken whenever it is necessary to fight against liver deficiency and hemorrhagic signs. One major problem is the treatment of renal disease that may require treatment.