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Characterization of circulating Leptospira strains in the French
overseas territories
Mathieu PICARDEAU
Unité de Biologie des Spirochètes
Institut Pasteur, Paris, France
The cycle of leptospirosis
Ko, Goarant, and Picardeau. 2010. NMR
Pathogens (9)
Intermediates (5)
Saprophytes (7)
The genus Leptospira: 21 species and >300 serovars
Neighbor-joining
phylogenetic trees
based on the analysis
of the 16S rRNA gene.
• Spirochete
9 Leptospira spp.
>250 serovars
• Most widespread zoonosis
• Survive in environment for weeks to months
• Penetrate abraded skin or mucous membranes
• Disseminate rapidly
• Colonize the renal tubules and shed in urine
The agents for Leptospirosis
• Affects the most neglected and
marginalized populations
• Limited information on disease
burden to guide effective policy
• Lack of adequate diagnostics
• No effective control measures
• Safe vaccines are not available
• Limited understanding of the
epidemiology and pathogenesis
Leptospirosis as a Neglected Disease
and an emerging disease
Demographic changes
• one billion individuals live
in slums
• the world’s urban slum
population will double in
the next 25 years
Climatic changes
• Global warming
• Extreme climatic events
(heavy rainfalls, etc)
Month of Hospitalization
Annual Rainfall-Associated Epidemics of Severe Leptospirosis: Population-Based Surveillance in Salvador, Brazil, 1996-2009 (N=2,607)
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
N o
. o
f m
o n
t h l y
c a
s e
s
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
M o
n t h
l y r
a i n f a
l l (
m m
)
N o . c a s e s
M o n t h l y r a i n f a l l ( m m )
Albert Ko, Yale university
>1,000,000 severe cases of leptospirosis /year mortality rate of 10%
Modeling leptospirosis incidence: work in progress (LERG)
?
Infection produces leptospiraemia in the first few days after exposure. Leptospires are then
cleared from the bloodstream as the titres of serum agglutinating antibodies increase (immune
phase). Leptospires are also transiently shed in urine for long periods.
immune phase (week 2)
PCR/culture from blood
IgM ELISA (months)
MAT (months/years)
Leptospiraemia (week 1) incubation period (5-14 days)
PCR/culture from urine (months)
D0: onset of symptoms
Diagnosis of leptospirosis
• symptoms mimick other febrile illnesses (malaria, dengue, etc)
• low sensitivity of diagnostic tests: PCR (blood, urine) serology (Microscopic Agglutination Test, ELISA)
Better knowledge of the epidemiology (reservoir, transmission, etc) Prevention measures Vaccines
Typical reservoir hosts
of common leptospiral serovars
• Microscopic Agglutination Test (MAT) on serum samples: serogroup (not always)
• Molecular and serological typing on culture isolates: species, serogroup, serovar, genotype
• DNA amplification from biological samples: species, genotype/serovar
Identification of the circulating agents
•To preserve and distribute reference strains, as well as
antisera, to other laboratories
•To identify leptospiral isolates and to contribute to
epidemiological investigations
•To participate in the training of scientists from other countries
in diagnostic techniques
•To develop research activities
The missions of the National Reference Center and WHO Collaborating Center for Leptospirosis (Institut Pasteur) :
World map with Mayotte and the French West Indies
Mayotte
French West Indies
• Culture isolation from patients and animals
• Direct amplification from blood (human) and kidneys (animals)
• Strain identification by serological and/or molecular techniques
(PFGE, MLST, MLVA, sequencing of 16S rRNA and secY)
Average no per year Average annual rate/100,000 inhabitants
Guadeloupe 2002-2008 99.4 22.5
Martinique 2002–2008 54.8 13.9 Guadeloupe 2011 267 [183–351] 69.4 [47.6–91.1] Martinique 2011 240 [144–337] 60.6 [36.3–85.0]
Burden of leptospirosis in 2002-2008 and results of the 2011 incidence study
French West indies, Caribbean sea
Martinique and Guadeloupe
• Common geological environments
• ∼400,000 inhabitants
• Similar levels of urbanization
• Economy depends on tourism and agriculture (sugar cane and bananas)
• The climate is tropical with a dry and reany season
Serovar diversity of pathogenic Leptospira circulating in the French West Indies
• Guadeloupe: 85 human isolates and 6 R. rattus isolates
• Martinique: 55 human isolates
• Detection of antibodies in patient sera by MAT
MAT is an unreliable predictor of infecting serogroups
(cross-reactions between serogroups)
Only culture isolation or DNA amplification / sequencing
allow definitive identification of infecting serovars or genotypes
13 genotypes (5 species, including L. kmetyi)
serovars Icterohaemorrhagiae/Copenhageni (30%)
serovar Arborea (20%)
serovar Bogvere (20%)
serovars Tabaquite and Bajan and other unknown serovars
Rodents, opossums, mongoose, bats, pigs, cattle, and dogs are hosts of pathogenic Leptospira species. •Serovars Icterohaemorrhagiae and Bogvere were isolated from rats, mice, and mongoose •Serogroup Ballum was isolated from rats and mice •Serovar Bajan was isolated from toads and frogs in Barbados
Animals responsible for the transmission of the disease
201100514
201003952
200701401
200309420
200305347
200305252
200309419
Bogvere
rat
isolates
human
isolates
Pulsed Field Gel Electrophoresis
Bim (75%) Icterohaemorrhagiae Arborea Bajan no Bogvere
Bogvere (35%) Arborea Icterohaemorrhagiae no Bim
Icterohaemorrhagiae (35%) Arborea Bogvere no Bim
Bogvere
Tabaquite
Circulating strains in the Caribbean islands
• Different distribution of the predominant pathogenic leptospiral serovars • Correlation with distribution of the animal reservoirs in these islands ?
100 miles
•200,000 inhabitants • 53% <20 years old • unemployment is high • 25% immigrants from the Comoro Islands • most households lack proper sanitation • proximity with animals (dogs, sheep, cattle, or goats)
Mayotte, Indian Ocean
Monthly rainfall (in mm) and number of cases of leptospirosis, 2009-2011
• age 13-78 years (mean 32.1)
• males represent 73.4% of cases
• 91% of the cases occured during the reany season
• 11 patients have been admitted in intensive care unit
• 3 patients did not survive
Cumulative incidence 2009-2011 of leptospirosis cases per commune of residence
Isolates from 94 patients (2007-2010)
Mini
Pomona
Pyrogenes/Ballum
Mini
Pyrogenes
Mini/Hebdomadis
Grippotyphosa
Grippotyphosa Mini/Hebdomadis
L. borgpetersenii
new species
L. interrogans
L. kirschneri
1%
Phylogenetic relationships of leptospirosis isolates based on MultiLocus Sequence
Typing (MLST) gene sequences (adk, lipL32, secY, rrs2, and lipL41)
16 sequence types (ST)
identification of a new species (confirmed by genome sequencing)
serogroup Mini is predominant (>70%)
serogroup Icterohaemorrhagiae is not present
species ST serogroup
Molecular and serological features of Leptospira isolates
L.interrogans
L.santarosai
L.borgpetersenii
L.kirschneri
Strains of Mayotte are colored as follows:
L. interrogans
L. borgpetersenii
New species
L. kirschneri
L.weilli
Minimum Spanning Tree of 372 Leptospira strains, including strains from Mayotte and previously
described strains (Nalam et al. 2010)
MLST
None of the STs from Mayotte strains have been previously defined
Leptospira serogroup and species distribution in animals
A total of 292 animals were sampled between march-may 2007: 156 rats, 50 brown lemurs, 49 flying foxes, 29 domestic dogs, and 8 stray dogs Similar distribution of species and serogroup between humans and animals
Risk factors: •Sex (males) •Age •Living in Comoros islands >10 years •Contact with animals (goats, zebus) •Proximity to water source or open sewers •Poor housing (floor type) •Contact with soil/water •Farmers/unemployed
• Blood samples were collected from 1414 participants in March 2011
• Questionnaire data were used to explore associations between
seropositivity and risk factors (demographics, and exposures at home, work, and
during recreation)
• 14.9% of participants were seropositive
• Global seroprevalence in Mayotte: 16.5% (IC95% 13.6-19.7) • Seroprevalence in La Réunion <1% in 2006 (Desvars et al. 2011)
• Serogroups: Mini (74.7%), then Pyrogenes, Grippotyphosa, Pomona, etc
Seroprevalence study in Mayotte, 2011
Distribution of seropositive cases
• Underestimation of the disease (better surveillance > higher incidence)
• Underestimation of the diversity of serovars and genotypes (new serovars/ genotypes/ species)
• High diversity of reservoirs
• No strict association between serovar and reservoir
• Similar distribution of serovars between humans/animals (not
only in rodents)
• Circulating strains in Mayotte differs from what is observed in
other countries (including La Réunion island)
Epidemiology of leptospirosis in Mayotte, Martinique, and Guadeloupe
The epidemiology of leptospirosis remains poorly understood
• Mode of transmission (environmental and behaviour risk
factors)
• Ecology of the bacterium (survival in the environment,
virulence, etc)
• Factors responsible for host specificity
• Circulation of strains between neighboring countries
(history of colonization of animal reservoirs in these islands)
Development of new diagnostic tools
Poor sensitivity of standard diagnostic test
MAT is a fastidious and complicated technique with low epidemiological value
Isolation and identification of circulating strains
Better knowledge of the epidemiology (serovar/animal reservoir) and ecology
Efficient diagnostic tools
Prevention measures (control of animal population, vaccination, etc)
Vaccine development
Current animal and human vaccines confer short-term efficacy and serovar-specific
immunity
Virulence factors of leptospires and pathogenesis
Infectious process poorly understood
Clinical investigations
Evaluation of prognostic factors for severe cases (serovars responsible for more
severe diseases ?)
Modeling
Disease transmission (animal population, environmental factors, etc)
Challenges
Amélie Desvars (animal study) Tinne Lernout (seroprevalence and incidence studies) Louis Collet (culture isolation of human isolates)
Sylvie Cassadou (incidence study) Patrick Hochedez (leptospirosis in Martinique)
The « Biology of Spirochetes » Unit The National Reference Center and WHOcc for Leptospirosis
Ambroise Lambert
Nadia Benaroudj
Pascale Bourhy
Annie Landier
Sylvie Brémont
Farida Zinini
Azad Eshghi
Christopher Pappas
Acknowlegments