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)

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M o

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m m

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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