ESCMID Postgraduate Technical Workshop Intracellular : from biology to clinic Villars-sur-Ollon, 26-30 August 2013 Our invisible neighbors

Rickettsiae around the world  XVII century: van Leeuwenhoek invents the microscope  XIX century: Cohn & Koch, founders of bacteriology  1878: Sedillot invents the term «microbe»  1925: Chatton separates Procaryotes & Eucaryotes Pierre-Edouard Fournier (nucleus absent in prokaryotes)

Centre National de Référence des rickettsioses, de la fièvre Q et des bartonelloses

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Our invisible neighbors New technologies, new questions  1957: Lwoff defines viruses 1980: 1,791 validated bacterial species Size < 200 nm, 1 nucleic acid, no autonomous replication, strictly 16S rRNA sequencing intracellular, no energy metabolism 2013: >12,000 species (670% increase)

 1975: Woese classifies living organisms in 3 domains: Eukaryotes, Bacteria, Archae U U © byR author R

ESCMIDExpansion of the Online microbial world LectureAn unsuspected Library biodiversity

14000 4000 Candidatus ‘’Carsonella ruddii’’

3500 0.15 Mb 12000 bacterial species bacterial genomes 3000 10000 viral species 2500 8000 viral genomes 2000 6000 1500 4000 1000 Ktedonobacter racemifer 2000 500 13.662 Mb 0 0

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1 Genomics => redefinition What about intracellular bacteria ?

of life boundaries st Koonin. Virology: Gulliver among the Lilliputians.  1909: Ricketts 1 describes a pathogenic Curr Biol. 2005 Mar 8;15(5):R167-9 intracellular bacterium that replicates autonomously and causes Rocky Mountain

Viruses => 1.2 Mb

Prokaryotes (0.15 – 13 Mb)

Dermacentor andersonii Eukaryotes (0.5 Mb => )  Major pathogens majeurs: C. burnetii, C. trachomatis, M. leprae, R. prowazekii

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R. rickettsii rickettsii R. prowazekii R. prowazekii R. tsutsugamushi R. quintana E. ewingii Puzzling bacteria C. burnetii E. canis E. ruminantium E. canis Anaplasma phagocytophilum E. phagocytophila A. marginale  1970s E. sennetsu Wolbachia pipientis C. ruminantium N. helminthoeca sennetsu  Few culture systems N.helminthoeca “No respectBartonella quintana for B. henselae W. pipientis  W. persica ”B. talpae  Few phenotypic characteristics B. bacilliformis R. grylli Brucella melitensis  Any intracellular bacterium, including B. bacilliformis Rickettsiella grylli G. talpae  Chlamydiae, is a “rickettsia” «Wolbachia persica» A. marginale E. ovis Eperythrozoon ovis Hemobartonella felis  Pathogenic species are all known (Rickettsia: H. felis Mycoplasma pneumoniae Gram + U 7 species in 1984, 17 in 2013)© byR author Ureaplasma urealyticum

What is a rickettsia? ESCMID Online LectureGenus Rickettsia Library Genus Rickettsia

 -  Gram-negative bacteria  Associated to arthropods:  Stained by the Gimenez , , lice, method  26 validated species, incl. 17 pathogens  Size 0.8 – 2 x 0.3 µ  > 100 unclassified Rickettsia sp.  Strictly intracellular  Major human pathogens  Associated to endothelial  B List of potential bioterrorism agents cells

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2 One -borne rickettsioses in 2013 per continent

R. massiliae R. conorii caspia

R. sibirica R. sibirica R. parkerii R. helvetica sibirica mongolitimonae

R. mongolitimonae

R. conorii indica R.slovaca R. conorii conorii R. conorii R. japonica israelensis

R. rickettsii R. conorii conorii

R. australis R. aeschlimannii R. africae R. honei

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Few phenotypic characteristics

Spotted fever group group - Associated to ticks - Lice-fleas Taxonomy -Spotted fevers - Typhus - Motile - Non motile - Optimal growth temperature 32°C - 35°C - Cross-reaction with strain OX-2 -with OX-19 U U © byR author R

ESCMIDPhenotypic characteristicsOnline LecturePhenotypic characteristics Library Complement fixation Plotz 1944, Pickens 1965  Mouse serotyping Philip 1978

Toxinic neutralization Bell 1960  Western-blot (rOmpA & rOmpB) Gilmore 1991

Proteic profiles (SDS-PAGE) Eisemann 1976, Pedersen 1978  Monoclonal antibodies Anacker 1987

R. conorii 7 R. conorii M R. honei R. massiliae R. akari R. felis R. australis R. montanensis R. japonica R. rickettsii R. canadensis R. typhi 2% R. prowazekii (Fang R. CDLI 2003) U Fang R. CDLI 2003 U R R

3 Antibiotic susceptibility Erythromycin Rifampin Genomic characteristics RSR. sibirica RSR. africae RSR. conorii RSR. slovaca RSR. honei Species GC % DNA-DNA Hybridization RSR. rickettsii R. rickettsii R. prowazekii RSR. japonica R R Bar 29 R. conorii 32-33 91-94% - R R R. massiliae R R R. rhipicephali R. rickettsii 32-33 - 47% R R R. aeschlimanii R. sibirica 32-33 70-74% - R R R. montana R. akari 32-33 46%- RSR. helvetica RSR. australis R. canadensis 29-30 47% 45% RSR. akari S S R. typhi R. prowazekii 29-30 47%- S S R. prowazekii R. typhi 29-30 42% 72% RSR. canadensis

RSR. bellii U U R R Rolain et al. AAC 1998;42:1537-41

Overall Taxonomy Official criteria for defining bacterial  Few phenotypic characteristics species are not adequate  Few genetic studies  Bacterial species = DNA-DNA hybridization: > 70% +  Poorly reproducible discriminatory and comparable G+C content: divergence < 5% + 16S rRNA: divergence tests < 1.3%

 DNA-DNA hybridization: R. montanensis, R. rickettsii, R. sibirica et R. conorii = same species -G+C content: SFG32-33%, TG 29-30%  Bergey’s manual 1984: 10 validated species - 16S rRNA: divergence < 2% among Rickettsia  7 pathogenic => The Rickettsia genus would only comprise

 Pathogenic vs non pathogenic U 3 species (vs 26 in 2013) U © byR author R

New bacterium

Taxonomic recommandations rrs > 98.1% with at least one AND validated Rickettsia species ESCMIDfor classifying rickettsial isolates OnlinegltA > 86.5% Lecture Library No Yes Other genus Member of the Rickettsia genus

> 1 of these criteria Fournier et al. J.Clin.Microbiol.2003;41:5456-65 rrs > 99.8% gltA > 99.9% with a validated Raoult et al. Ann.NY.Acad.Sci.2005;1063:232-43 ompA > 98.8% ompB > 99.2% Rickettsia species sca4 > 99.3%

Yes No Belongs to a Potential new species Rickettsioses validated species

Isolated in pure culture Isolated in pure culture Yes No Yes (molecular data only or isolate lost upon subcloning) SPD > 3 with respect Describe to the type strain phenotypic characters AND (geographic distribution, Specific epidemio- clinical vector, pathogenicity (if any), characteristics Strain of Candidatus mouse serotype with the species respect to genetically Emerging zoonoses closest validated species) Yes Subspecies

Deposit of the type strain into two independent official culture collections AND publication in Int. J. Syst. Evol. Microbiol. or publication in another peer-reviewed journal and in a Validation list in Int. J. Syst. Evol. Microbiol. U U No Yes R R Unvalidated "species" or "subspecies" Validated species or subspecies

4 Tick hypostoma inserted in the skin of a patient

Tick-borne rickettsioses

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Europe 2012: emergent pathogens

Europe and Mediterranean area

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ESCMID Online LectureMediterranean Spotted Library fever (MSF)

One of the oldest recognized vector- borne infectious diseases

MEDITERRANEAN SPOTTED FEVER 1909: first cases in (Conor and Brush) 1925: first description of the eschar in Marseille (Boinet and Pieri)

Rickettsia conorii subsp. conorii 1930’s: - identification of the vector: (Olmer – Durand & Conseil)

- identification of the agent : R. conorii (Brumpt)

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5 Global distribution of MSF/ Rh. sanguineus R. conorii conorii – Rh. sanguineus

 2009: 100% vertical transmission of R. conorii in naturally infected Rh. http://jfbradu.free.fr/cartesvect/fdcmonde.htm sanguineus over 12 generations BUT  Prevalence of infected ticks in the wild R. conorii detected in the <1%, with exception in endemic region: tick ovaries by electronic microscopy small foci? – Effect of climatic conditions on survival of infected tick

Reactivation phenomenon for R. conorii => tick lethality Many gaps in the Ecology of MSF: Rh. sanguineus has a worldwide distribution but Socolovschi et al. 2009; Socolovschi et al. 2012 MSF is known to be only endemic in Mediterranean area

Rickettsia c. conorii –animal reservoir? Mediterranean spotted fever : clinical aspects

Which host for Rh. sanguineus ? • Urban disease 2/3 - rural 1/3 Peak • Tick-bite is often unnoticed (larva, nymphs++) in August • Seasonal zoonosis: warmest period • Males • Incidence 50/100 000 hbts

• Incubation 6 – 7 days • Fever 100%, flu-like symptoms • Inoculation eschar (single ?) 72 % • Conjunctivitis

• Skin rash 97%

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MSF rash May occur at any age ESCMIDinvolves palms-soles, Online not face Lecture Library

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6 Evolution Changing epidemiology of MSF

 Most often mild !!! Global warming => effect on tick  6% => malignant behavior forms • ↑ period of activity of Rh. Sanguineus • ↑ aggressiveness  Diabetes, • ↑ biting of unusual hosts (humans) immunodeficiency, => Multiple eschars, severe forms Fatal MSF French in G6PD deficiency, => ↑ incidence of Rh. sanguineus-transmitted 2003 heat wave: 22 attached ticks elderly, alcoholism, diseases on a man delayed antibiotics

 Mortality 2-3% Fatal cases 2 – 6%, and reach 30% in hospitalized patients Multiple eschars Mouffok et al. Int J Infect Dis 2009; 13: 225-37 Socolovschi et al. 2009. Parola et al. 2008

Other subspecies

• R. conorii subsp. israelensis • Israeli spotted fever

• Sicily, Portugal: • Rh. sanguineus ticks, Other Rickettsia conorii subspecies • Human cases: Inoculation eschar 4% • Imported fatal cases: UK (from Portugal), Switzerland (from Lybia) Rickettsia conorii subsp. israelensis • R. conorii subsp. caspia Rickettsia conorii subsp. caspiensis • Astrakhan fever Rickettsia conorii subsp. indica • Kosovo, France: Rh. sanguineus ticks (cluster of spotted fever) • No human cases in Europe • Imported cases: France (from Chad)

• R. conorii subsp. indica • Indian tick typhus • Sicily : one human case • Imported cases: French traveler (from India) U U R Chai JT JTM 2008; Boillat et al. 2008; Levin et al. 2012; Alexandre N et al.2011; Tarasevich 1991; Parola et al. R © by author2001; Renvoisé et al. 2012

ESCMID Online LectureSENLAT - TIBOLA - DEBONEL Library

 1997: R. slovaca as human pathogen (Lakos. Lancet. 1997) (scalp eschar + enlarged cervical lymph nodes)

 2002: Lakos proposed the name of this syndrome SENLAT TIBOLA (TIck-BOrne LymphAdenopathy) (SCALP ESCHAR AND NECK LYMPHADENOPATHY)  2006: R. raoultii = 2nd agent of this syndrome and Ibarra et al. proposed the name DEBONEL (Dermacentor-Borne Necrosis Erythema Rickettsia slovaca, , Rickettsia « rioja » Lymphadenopathy)

 2010 : Identification of 3 patients with henseae infection Raoult proposed the name SENLAT (Scalp Eschar and Neck LymphAdenopaThy)

U R Raoult et al. 1997; Lakos et al. 2002; Ibarra et al. 2006; Angelakis et al. 2010; Parola et al. 2009

7 SENLAT R. slovaca and R. raoultii infection

• Endemic: Europe

• Vecteur: Dermacentor marginatus and D. reticulatus

• The peak incidence: March-May & September- November

U R Parola et al. 2009 (Parola et al. 2009)

Unusual agents of SENLAT

• : 3 patients, France • Francisella tularensis : 1 patient, France LAR • Rickettsia “rioja”: 1 patient, Spain (Lymphangitis-associated rickettsiosis)

• Co-infections: mongolitimonae • R. slovaca-Coxiella burnetii: 1 patient, France

• R. slovaca-Borrelia burgdorferi : 8 patients, Spain

U U Lakos et al. 2002; , Parola et al. 2009; Angelakis et al. 2010; Edouard et al. 2012; Perez-Perez© et al. 2009 byR author R

ESCMIDR. sibirica mongolitimonaeOnline LectureR. sibirica mongolitimonae Library • Mediterranean area (France, 1996: 1st human case, Greece, Portugal, Spain) France, tick vector? • Potential vector in Europe: >20 cases to date • Hyalomma anatolicum 1991: Detection in Hyalomma excavatum (Greece, Cyprus) 2005: first case asiaticum, Mongolia • Rh. pusillus (France, in Algeria

8 Portugal) 7 R. sibirica mongolitimonae in ticks 6 5 2004: 4 Detection in H. truncatum 3 Human infection First case in 2 Niger 1 South Africa 0 Imported case: French traveler (from Egypt)

Migratory birds? France Greece Portugal Spain U U R R Raoult et al. 1996; Pretorius et al. 2004 Psaroulaki et al. 2005; Chochlakis D. et al. 2012; de Sousa et al; 2006; Edouard et al. 2012

8 LAR (Lymphangitis-associated rickettsiosis)

• Men 70%

• Fever 100% • Headache and myalgia: 87%

• Skin rash 90% Others spotted fever rickettsioses • Inoculation eschar: 87%, including multiple eschars 15% R. massiliae, R. monacensis, R. aeschlimannii, R. helvetica

• Lymphangitis expanding from eschar 32%

• Painful enlarged lymph nodes 65%

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Rickettsia massiliae

• 1992: first isolated from Rh. • 2002 : first isolated from Ixodes sanguineus and later from other ricinus ticks collected in Germany Rhipicepalus spp. in Europe, Africa, America • Infection rate in ticks: 2.4 % in Spain to 52.9% in Bulgaria • Infection rate in ticks: up to 92% in France R. massiliae in ticks Human infection R. monacensis in ticks • 2006: Identification of a SFG rickettsia isolated from blood in a Human infection Sicilian patient in 1984 as R. massiliae • 2008: 2nd patient in France • 2007: identification from blood in 2 patients from Spain • 2010: 3rd patient in Argentina • 2012: 1 patient in Sardinia

Vitale et al. 2005, Parola et al. 2008; Garcia-Garcia et al. 2010; Socolovschi et al. 2012 U U © byR authorMadeddu et al. 2012 R

ESCMIDRickettsia aeschlimanniiOnline LectureRickettsia helveticaLibrary • 1992: first isolated from Hyalomma • 1979: 1st isolation from I. ricinus 17 marginatum marginatum ticks from ticks from Switzerland Morocco

• Infection rate in ticks: 64.7% in • Several infection cases reported Corsica (H. m. rufipes) in Europe and Asia, but no isolation of the bacterium from • Transported through Europe by clinical samples migratory birds (France, Germany, R. hevetica in ticks Spain) R. aeschlimannii in ticks

• 2002: 1st human case, a French traveller to Morocco with MSF-like illness => presumably associated with human illnesses • No autochtonous case in Europe U U Raoult et al. 2002; Fernandez-Soto et al. 2003; Rumer et al. 2011; Socolovschi et al. 2012; R R

9 R. africae

(Raoult et al. N.Engl.J.Med. 2001;344:1504-10)

 Distribution of R. africae similar to that of Amblyomma

Africa  Sub-saharan Africa (Parola et al. N.Engl.J.Med.1998;338:1391; Parola et al. Am.J.Trop.Med.Hyg.1999;60:888-93; Kelly et al. 2003;unpublished data)

 Prevalence = 1,7 - 72% (Tissot-Dupont et al. Am.J.Trop.Med.Hyg. 1994;50:373-80; Beati et al. J.Med.Entomol. 1995;32: 787-92; Clin.Infect.Dis.1995;21:1126-33 Parola et al. Emerg.Infect.Dis. 2001;7:1014-17)

 Seroprevalence = 27 – 52% (Tissot-Dupont et al. Clin.Infect.Dis.1995;21:1126-33)  Most frequent SF rickettsiosis  Importation of Amblyomma

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Epidemiologie Clinical manifestations Raoult et al. N.Engl.J.Med. 2001;344:1504-10) (Raoult et al. N.Engl.J.Med. 2001;344:1504-10)

Amblyomma : attack strategy (CO2) • Sex-ratio H/F 1.6, mean age 45 years • Silent incubation, 6-7 days • Sudden onset, fever 88%

• Rash 46% = MP 51%, vesicular 45%, purpuric 4% grouped cases (74%) multiple eschars U U attack rate 4-14% © byR author R

Clinical manifestations Distribution of R. Africae in West Indies Raoult et al. N.Engl.J.Med. 2001;344:1504-10) ESCMID Online Lecture LibraryParola P, et al. N Engl J Med. 1998

• Inoculation eschar 95%, multiple 54%, limbs 73%

• Satellite enlarged lymph nodes 43% + Reunion island, New Caledonia • Mild disease (recovery 100%, no sequellae) U U R R

10 Rocky Mountain Spotted Fever

• • Vector = Dermacentor andersoni • summer America • USA : 500 cases/year (south-east) • central America, Brazil, Argentina, Colombia • Fever, rash, no eschar 1 - 2.5 p. 106 • Mortality 5 - 80 % 2.6 - 5 p. 106 without treatment > 5 p. 106

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R. parkeri R. parkeri

• 1st isolation in Amblyoma maculatum in 1931 • Prevalent tick in south-eastern USA • One isolate in a patient dead from “RMSF” in Ohio

partially similar to R. parkeri (Ralph et al. Ann N Y Acad Sci -1990) • Patients in Florida, Uruguay, Argentina • Fever, rash, eschar, R. parkeri isolated from a skin

biopsy (C. Paddock, CID, 2004)

Raoult D, Paddock C. N.Engl.J.Med. 2005: 626-7. U U © byR author R

ESCMID Online Lecture RasLibraryh Enlarged Disease Rash specificity Eschar lymph nodes Rocky Mountain spotted 90 % 45 % purpuric very rare no fever Mediterranean spotted fever 97 % 10 % purpuric 72 % rare Siberian tick typhus 100% 77 % yes Other tick-borne 100 % vesicular 65 % yes Israëli spotted fever 100 % rare no Flinder’s Islands spotted 85 % 8 % purpuric 28% yes fever rickettsioses Astrakhan fever 100 % 23 % no African tick-bite fever 30 % vesicular 100 % yes multiple 100 % 90 % no Rickettsia mongolotimonae yes yes no Rickettsia slovaca no yes yes Rickettsia helvetica no - no no R. heilongjangensis yes yes yes R. aeschlimannii yes - Yes no (multiple?) R. parkeri yes - yes ?

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11 R. aeschlimannii R. prowazekii Diagnostic tricks R. africae R. asiatica R. australis R. felis R. bellii R. typhi  Triad fever – eschar – rash R. conorii complex R. africae ? Human body R. canadensis R. heilongjiangensis louse R. honei Ixodidae R. hoogstraalii tick But R. helvetica R. japonica Other potential R. massiliae R. monacensis arthropod vectors Whitefly R. montanensis Rickettsia sp. MEAM R. parkeri ? Absence of characteristic symptoms R. peackockii R. philipii 364D No rash (SENLAT) R. raoultii R. rhipicephali Neither rash nor eschar (R. helvetica) R. rickettsii R. sibirica complex R. slovaca Mosquito R. tamurae Argasidae Mesostigmatid R. felis ? R. felis Coexistence of several rickettsioses in an R. prowazekii? tick R. hoogstraalii Booklice mites U Rickettsia sp. AvBat R. akari area R R. felis Mehrej et al. 2013 R. felis? R. felis

Acknowledgements : Pr. Didier Raoult Pr. Philippe Parola

THANK YOU ! © by author ESCMID Online Lecture Library

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