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Home , African tick bite fever, North Asian tick typhus, Queensland tick typhus, Rickettsialpox, Rickettsia africae, Rickettsia australis

Educational Workshop

EW14: Pathogenic intracellular arranged with ESCAR (ESCMID Study Group for Coxiella, Anaplasma, and )

Convenors: Gilbert Greub (Lausanne, CH) Pierre-Edouard Fournier (Marseille, FR)

Faculty: Amel Omezzine Letaief (Sousse, TN) Pierre-Edouard Fournier (Marseille, FR) Achilleas Gikas (Heraklion, GR) Gilbert Greub (Lausanne, CH)

Letaief –

Tick-Borne rickettsial Spotted Fever Group Rickettsioses (SFGR)

Amel Omezzine Letaief, MD Farhat Hached Hospital ; Sousse -

22/04/2009 ESCAR, 19th ECCMID - Helsinki 1

Session objectives ? True or False ?

•There are multiple SFGR •SFGR are of limited geographic distribution •Clinical features are specific of rickettsia species •SFGR are severe diseases •Diagnosis of SFGR is difficult to confirm •Therapy of SFGR has been optimized

22/04/2009 ESCAR, 19th ECCMID - Helsinki 2

Clinical case

• A 55-year-old Finnish man, traveled to Africa and south Europe : Cruise on the for 10 days, on September • 5 days after return : Abrupt onset of fever (40°C) Loss of appetite, Headache Abdominal pain 1 episode of diarrheae

• No medical history, no

22/04/2009 ESCAR, 19th ECCMID - Helsinki 3

3 Letaief –Spotted fever rickettsiosis

Clinical case Continues… ______•Physical examination at day 3 : –No signs of severe sepsis –Fever + generalized maculo-papular –Purpuric lesions on legs - Biological tests including : –blood smears (-), –blood cell count, normal •And Rickettsioses!!… SFGR ?

22/04/2009 ESCAR, 19th ECCMID - Helsinki 4

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Which Rickettsia ?

Order : et Bartonellaceae (Bartonella + Rochalimaea)

Tribu : Anasplasmataceae

Genus : Orientia Rickettsia

O tsutsugamushi group (Spotted Fever Group) ()

Epidemic typhus R.prowazekii R rickettsii RMSF R.typhi R. conorii MSF R. akari vesicular fever R. africae African bite rickett Excluded from Rickettsiales : R. felis - typhus () …. 22/04/2009 ESCAR, 19th ECCMID - Helsinki 6

4 Letaief –Spotted fever rickettsiosis

SFG Rickettsial pathogens and diseases

• • Flea-borne spotted fever or cat flea typhus • • Rickettsia helvetica Aneruptive fever • Spotted fever • Rickettsia aeschlimannii Spotted fever • Rocky mountain spotted fever • Rickettsia sibiricasibirica Siberian tick typhus • Rickettsia sibiricamongolitimonae LAR • Rickettsia slovaca TIBOLA-DEBONEL • • conorii Mediterranean spotted fever • Rickettsia conorii israelensis Israeli spotted fever • Rickettsia conorii caspia Astrakhan fever • Rickettsia conorii indica Indian tick bite typhus • Rickettsia heilongjiangensis Far Eastern tick-borne rickettsiosis • Japanese or oriental spotted fever • Spotted fever • Flinders island spotted fever • DEBONEL-TIBOLA 22/04/2009 ESCAR, 19th ECCMID - Helsinki 7 A. Renvoisé, D. Raoult / Médecine et maladies infectieuses 39 (2009)

SFGR

• Most tick - borne rickettsioses !!!! •R. akari (Ricketsialpox) : •R. felis : flea • Disease named by geographic distribution •Mediterranean spotted fever •Rocky Mountain spotted fever •Queensland tick typhus •. • Human are incidental host • Natural host are small mammal !! Not all known •wild , mouse, dog

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Life cycle- Human contamination

Human yes

TICK : no non Adult Vector and Adult♀ ♂ Reservoir

ouiyes Nymph

larva vertebrate Host ? reservoir?

22/04/2009 ESCAR, 19th ECCMID - Helsinki 9

5 Letaief –Spotted fever rickettsiosis

~ 20 SFGRickettsial pathogens…

R. C. Astrakhan R. slovaca R. mongolotimonae R. helvetica R. sibirica

R. mongolotimonae R. C israëli R. helvetica R. conorii R. aeschlimannii R. C israëli R. japonica R. conorii R. rickettsii R. C. indica R. conorii

R. australis R. africae R. honei

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Distribution, vector, and main clinical features of the different subspecies of SFGR

Symptoms , % patients Fatal forms? Rickettsia Vector tick Geographic repartition Human Disease Inoculation (% patients) Fever Rash R. conorii Rhipicephalus Mediterraneanarea Mediterranean 91– 20–87 93–100 Yes conorii, sp., Croatia, Slovenia, , spotted fever 100 (0–18.1) isolates Haemaphysali Somalia, South Africa, Malish, s leachii and surrounding the Moroccan Black Sea Kenyan R. conorii Rh. Israel, Portugal, Sicily Israeli spotted 100 0–46 98–100 Yes israelensis sanguineus fever (0–3. 5)

R. conorii Rh. Astrakhan region, Chad, Astrakhan 100 23 94 No caspia sanguineus, R. Kosovo pumilio spotted fever

R. conorii Rh. India, Pakistan Indian tick 100 Rare 100 No indica Sanguineus typhus (freq. Boophilus purpuric) microplus, H. leachii R. rickettsii North, Centerand south Rocky 100 Rrare 100 Yes, 5-10 1919 andersoni America mountain purpuric spotted fever

22/04/2009 ESCAR, 19th ECCMID - Helsinkiadapted from ww.cdc.gov/eid• Vol. 14, No. 9,11 2008

Distribution, vector, and main clinical features of the different subspecies of SFGR

Symptoms , % patients Fatal Geographic Rickettsia Vector tick Human Disease forms? repartition Inoculation Fever Eschar Rash (%) R. Slovaca Dermacentor Europe TIBOLA - rare >80, drai- No No marginatus DEBONEL ning ADP

R.africae Amblyomma ; Souh Africa, west African tick-bite 88 Yes (55), 50 No Indies fever multiple R. Aeschli- Hyalomma Africa, South Europe Spotted fever 100 Yes, multiple yes No mannii marginatum (rare cases reported)

R. akari mite USA, Corea, East Rickettsialpox 97- 90 100 No Europe 100 vesicular

R. japonica Haemophysalis Japon, Chine Japanese/ Oriental 100 90 purpiric Severe Dermacentor spotted fever forms up to20% (4) R. sibirica Dermacentor Central Asia, China Siberian tick yes yes yes ? Haemophysalis typhus R. mongo- Hyalomma China, Europe, Spotted fever yes yes Few spots No lotimonae asiaticum Africa

R. australis Ixodes Australia Queensland tick yes 50% Yes low holocyclus fever vesicular R. honei Ixodes, Thailand, Australia Flinders Island tick yes rare Yes, ? Aponomma typhus purpuric 22/04/2009 ESCAR, 19th ECCMID - Helsinki 12

6 Letaief –Spotted fever rickettsiosis

Epidemiologic and Clinical Features frequent SFGR

• Mediterranean Spotted Fever Group (MSF) R.conorii – Hot season – Mediterranean region, south Europ, Africa – Rural area, dogs tick –bite – Eschar inoculation « Tache noire » + fever + Maculo-papular eruption – Rare severe forms • Rocky Mountain Spotted Fever (RMSF) R. rickettsii – America, North – Tick bite, – Fever + purpuric rash, eschar rarely – Frequent severe forms

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Epidemiologic and Clinical Features frequent SFGR

• African tick-bite fever R. africae – Hot season – Africa, subsaharian – multiple tick –bite – of inoculation + fever + Maculo-papular eruption – Rare severe forms • TIBOLA : Tick-Borne Lyphadenopathy R.slovaca – Europe – Tick bite, winter, women and children!! – Eschar of the scalp + Fever + Lyphadenopathy – Sequellae : alopecia

22/04/2009 ESCAR, 19th ECCMID - Helsinki 14

Mediterranean Spotted Fever History

– 1910 : disease described by CONOR and BRUSH in Tunisia – 1925 : «tache noire» described by PIERI in Marseille – 1930 : role of dog’sTick – 1932 : R. conorii by Brumpt

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7 Letaief –Spotted fever rickettsiosis

MSF Clinical features of hospitalized patients

Central Tunisia Marseille Clinical findings (%) (%) Fever (100) (100) Headache (76) (56) The clinical triad Chills (46.5) - fever +rash+ Eschar Cough (17.5) (10) 55 - 70% Myalgia (58) (36) Rash (92.5) (97) Eschar lesion (62) (72) Conjunctivitis (12) (9) Gastrointestinal symptoms (20) Hepatomegaly and or (8) (10) splenomegaly

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Characteristics of eschar (Tache noire)

Central Tunisia (%)

Presence of Eschar (62)

Number one (95.5) 2 (4.5) Sites of Eschar trunk (16) extremities (33.5) gyrus (9.5) genital organs (24.5) breast (4) others (12.5)

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8 Letaief –Spotted fever rickettsiosis

Laboratory findings

Central Tunisia Marseille (%) (%)

Leukopenia and/or (79) (75) normal blood cells (55) (35)

Elevation of (60) (39) transaminases

Hyponatremia (58) (25)

Increased LDH level (94) -

Creatinine (µmol) >150 (5) (6)

Elevated ESR (> (80) - 30mm/h)

ESR: eleva ted erythrocyte sedi mentation rate 22/04/2009 ESCAR, 19th ECCMID - Helsinki 19

Ocular manifestations

• Prospective study, 30 patients with MSF underwent complete ophthalmic examination • 25 (83.3%) had unilateral or bilateral posterior segment involvement (white retinal lesions), 9 patients had ocular complaints • Final visual acuity was 20/20 in 93% of affected eyes

Khairallah M, Ophthalmology 2004; 111 (3): 529-34

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Diagnosis Score

Epidemiologic criteria • Endemic area 2 • Hot season (May –September) 2 • Tick bite 2 ≥ 17 Clinical criteria S=85% • Fever exceeding 39°C 5 SPF=30% • Eschar (tache noire) 5 • Maculopapularor purpuricrash 5 • Two (of three) clinical criteria 3 • All three clinical criteria 5 _____ 29

Letaief A. Ann N Y Acad Sci. 2003 (from Raoult’sscore)

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9 Letaief –Spotted fever rickettsiosis

Evolution

• Natural history (without ) : Frenquently favourable 2–3 wks, long convalescence, Severe forms : 25-50% • Treated : Good prognosis : 5-10 days, – Apyrexia : 3-4 days, – Severe forms 3-18%

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Complications and malignant forms

Central Marseille Tunisia France %

Prevalence Severe forms 5 6 Rate of mortality 0 2.5

Mean age of patients with severe forms 63.5 years 65

Types of complications neurologic ++ ++ cardio vascular + ++ renal ++ ++ gastrointestinal 0 - others + -

Mean duration of hospitalization 8.1±2.7 - (days)

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RockyFIEVRE Mountain BOUTONNEUSE Spotted Fever MEDITERANEENNEHistory

• 1899, Maxey described RMSF “the Snake River Valley” of Idaho • Role of Dermacentor by King and Ricketts in 1906 • Ricketts isolated the causative organism in guinea pigs • 1916, Wolbach described R rickettsii using the Giemsa stain • In 1919, R rickettsii , intracellular , vasculitic lesion • In 1940s, and tetracyclines treat RMSF

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10 Letaief –Spotted fever rickettsiosis

RMSF Clinical features

(%) Clinical findings

Fever (100) Headache (85) Chills - Cough (33) Myalgia (70) Rash, (petechial 50%) (90) Eschar lesion (<1) Conjunctivitis (30) Gastrointestinal symptoms (40-60) Hepatomegaly and or splenomegaly (15)

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R rickettsii, RMSF

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African tick-bite fever - R. africae

• Sub-Saharan Africa, Caribbean • November –April , travelers • clinical signs generally mild, no deaths nor severe forms reported • Rash 50% • Multiple inoculation eschar 55% • tick bites 44% • Fever 88% • Eschars on lower limbs 62% • Enlargement of lymph nodes 43%.

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11 Letaief –Spotted fever rickettsiosis

TIBOLA- DEBONEL - R. slovaca

• TIBOLA TIck-BOrne

• DEBONEL DErmacentor-BOrne--Erythema-Lymphadenopathy • Early spring, autumn ; and winter in southern Europe • Women and Children * inoculation eschar particularly on the scalp * enlarged lymph nodes draining area * Fever and rash were uncommon, * Sequellae : localized alopecia at the bite site and chronic fatigue

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Dermacentor marginatus

22/04/2009 ESCAR, 19th ECCMID - Helsinki 29

Clinical case Continues… ______• Patient hospitalizedon day 5, because of –Severe sepsis –Hepatomegaly –Generalized rash, no eschar –Agitation,

• antibioticprescribed : Ceftriaxone 2 gr IV + Vibramycine 200 mg • Serologies made • Negative blood cultures

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12 Letaief –Spotted fever rickettsiosis

Results of serology rickettsoses

•At day 3 of apyrexia •1st serology: R.conorii (Ig G 1:64 ; Ig M 1: 128) •Seology 15 days after Ig G IgM –AB anti-R. conorii 1: 256 1:128 –AB anti-R. typhi 1: 256 1:64 –AB anti-R. felis 1: 128 1:64 –AB anti- R. aeschlimannii 1 : 512 1:256

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How to confirm SFGR

• Serology routinely • Culture referencelaboratory • Histochemicaland Immuno- rarely Histochemical methods • Molecular Tools : more frequently PCR and sequencing methods

22/04/2009 ESCAR, 19th ECCMID - Helsinki 32

Confirm SFGR SEROLOGY

• Reference standard : IFA detecting IgG et IgM •Sensitivity 5th – 9th day 46% 20th – 25th 90% insufficient to idenify the etiologic agent CA, Western Blot ++++

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13 Letaief –Spotted fever rickettsiosis

Confirm SFGR Molecular methods -PCR •Sensitive, rapid ++++ •Availability ± •Specimens –In Blood ± –In skin biopsy –Ticks , as epidemilogical tools

•Nested PCR

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How to treat SFGR

• Intra- cellular bacteria • Antibiotics :To be empirically prescribed in any SFGR, before confirmation of the diagnosis – Tetracyclines, ++ – – Chloramphenicol – Macrolides : Clarithromycin, Josamycin, – Rifampicine (in vitro)

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Antibiotic regimens MSF

• Adults

– Doxycycline * 2 oral 200-mg doses /12-h interval * 200 mg single dose or 100 mg twice a day, 2 to 5 days

– Josamycin * 2 oral doses of 1 g / 8 h for 5 days

– Ciprofloxacin * 750 mg every 12 h for 7 days • Children

– Doxycycline * 2.2 mg/kg /12 h <45 kg or adult dosage, 5 to 10 days

– Clarythromycin *15/mg/kg/day in two divided doses for 7 days

– Azythromicin, *10 mg/kg/day in one dose for 3 days

– Josamycin, * 50 mg/kg every 12 h for 5 days • Pregnant women * Josamycin, 50 mg/kg every 12 h for 5 days

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14 Letaief –Spotted fever rickettsiosis

Antibiotic regimens RMSF

• Adults

– Doxycycline * 100 mg every 12 h for 5 to 10 days • Children

– Doxycycline * 2.2 mg/kg /12 h <45 kg or adult dosage for 5 to 10 days – Chloramphenicol, * 12.5 to 25 mg/kg every 6 h for 5 to 10 days

• Pregnant women – Doxycycline * 100 mg every 12 h for 5 to 10 day

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Conclusion

• Many novel Rickettsia isolates, • A larger number of Rickettsia species are agents of human diseases • Spotted fever rickettsiosis syndromes are generally similar, not life threating, however –Malignant forms and mortality in RMSF, not only.. –Spotless form also, R. Slovaca, not only.. • Specific confirmation needs reference laboratory • Empirical antibiotic without delay +++

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15 16 Summary

Bartonella: from cat-scratch disease to endocarditis PE Fournier (Marseille, FR)

Bartonella species are fastidious gram-negative bacteria that belong to alpha- , which are associated with mammals. These bacteria are considered as emerging pathogens. They possess a natural cycle that involves persistent intra- erythrocytic infection in a reservoir host. vectors transmit the bacteria between reservoirs and disease-susceptible hosts, including humans. Of the nineteen officially validated species, seven are recognized human pathogens, including B. bacilliformis (the agent of or Carrion’s disease), B. quintana (which causes ), B. henselae (the agent of cat scratch disease [CSD]), B. alsatica, B. elizabethae, B. koehlerae, B. vinsonii subsp. arupensis, B. vinsonii subsp. berkhoffii, (agents of endocarditis), and B. grahamii (causing neuroretinitis). The variety of diseases caused by Bartonella sp. depends in part on the host immune and/or cardiovascular status. As an example, B. henselae can cause a focal suppurative reaction (CSD) in immunocompetent patients, a multifocal angioproliferative response () and/or a dilatation of hepatic sinusoids (peliosis hepatis) in immunocompromised patients, and an endovascular multiplication with development of endocarditis in patients with previous valvular defect. The natural history of complicated forms of B. henselae infections is not fully elucidated. However, the recent demonstration of the development of an endocarditis six months after CSD in a patient with known valvulopathy suggested that CSD may be B. henselae primo-infection. This also suggested that patients exposed to B. henselae may develop bacteraemia with or without, as observed in most patients with B. henselae endocarditis, clinical signs of typical CSD. Of these, patients with valvular lesions may subsequently develop endocarditis. As a consequence, because Bartonella endocarditis is a severe disease that often requires valvular replacement, we suggest that patients who develop CSD should be investigated for valvular defects, in which case they should be tested within a year for serological, blood culture or DNA detection of continued B. henselae infection.

17 18 Fournier - Bartonella

1919thth ECCMID, Helsinki 2009

BARTONELLA, FROM CAT SCRATCH DISEASE TO ENDOCARDITIS

PierrePierre--EdouardEdouard Fournier

Unité des Rickettsies et Pathogènes émergents URMITE, CNRSCNRS--IRDIRD UMR6236, Faculté de Médecine, Université de la Méditerranée Marseille, France U R

1919thth centurycentury Oroya fever, PeruPeru (Schultz. Am J trop Med hyg.Med hyg. thth 66 centurycentury 1968;17:5031968;17:503--15)15) PrePre--ColumbianColumbian huaca 18891889 20092009 representation of Parinaud’s 19501950 2121 Bartonella verruga peruana conjunctivitis Cat scratch species, incl. EcuadorEcuador (Parinaud. Ann diseasedisease 12 human (Alexander. Am J trop Med hyg. Occul.Occul. (Debre et al. Soc Med Trop 1995;52:3541995;52:354--9)9) 1889;101:2521889;101:252-- Paris. 1950;66:761950;66:76--9)9) pathogens 3)3)

thth WWIWWI 2,000 BC2,000 BC 1616 centurycentury 1990s1990s Trench fever B. quintana inin B. bacilliformis B. henselae Human teeth outbreaks in Spanish Byam et al. Oxford University Press. 1919 and B. FranceFrance soldierssoldiers quintana in PeruPeru bacillary angiomatosis and endocarditis Bartonella (Relman et al. N Engl J Med. 1990;323:15731990;323:1573--8080 (Drancourt et al. Clin Infect genusgenus Drancourt et al. N Engl J Med. Dis. 2005;191:6072005;191:607--11)11) (Strong et al. JAMA. 1995;332:4191995;332:419--23)23) (Alexander. Am J trop Med hyg. 1915;64:8061915;64:806--8)8) U 1995;52:3541995;52:354--9)9) R

Genus Bartonella

(Maurin M et al. Eur. J. Clin. Microb.Infect. Dis. 1997)

ØØ Aerobic, GramGram--negativenegative bacteria ØØ Stained with the Gimenez method

ØØ Size 0.5 ––0.6 x 1.0 µµ0.6 ØØ G+C%: 37 --4141 ØØ Facultative intracellular

ØØ Target cells = endothelial cells and erythrocytes

ØØ Highly fastidious ØØ Emerging pathogens

U R

1

19 Fournier - Bartonella

Taxonomy

ØØ aa--proteobacteriaproteobacteria ØØ 1984: Order Rickettsiales, family Rickettsiaceae (Bergey’s manual)

ØØ 1993: Unification of the genera BartonellaBartonella andand

Rochalimaea (Brenner et al. Int J Syst Bacteriol. 1993;43:7771993;43:777--86)86)

ØØ 1995: Unification of the genera Bartonella and

GrahamellaGrahamella (Birtles et al. Int J Syst Bacteriol.1995;45:1Bacteriol.1995;45:1--8)8)

ØØ 2009: 21 species

ØØ 3 subspecies in B. vinsoniivinsoniiB.

U R

a subsub--divisiondivision of Proteobacteria

Methanococcus spp. Archaebacteria Staphylococcus spp. Bacillus spp.

Streptococcus spp. Gram positive bacteria, low G+C% spp. spp. Bacteroides spp. Bacteroides-Cytophaga Fusobacterium spp. Fusobacteria Mycobacterium spp. Gram positive bacteria, highG+C% Borrelia spp. Spirochetes spp. Chlamydia Campylobacter spp. d-e

Brucella spp. a Bartonella spp.

Neisseria spp. b

Coxiella burnetii Proteobacteria Pseudomonas spp. Acinetobacter spp. g Vibrio spp. U Entérobacteriaceae R

A complex ecology

ØØ ZoonosesZoonoses =>=> ØØ HumansHumans ((BB..bacilliformis,bacilliformis, BB..quintanaquintana,, BB..elizabethaeelizabethae))

ØØ CatsCats ((BB..henselae,henselae, BB..clarridgeiae,clarridgeiae, BB..koehlerae,koehlerae, BB..bovis,bovis, BB..quintanaquintana))

ØØ DogsDogs ((BB..vinsonii,vinsonii, BB..quintana,quintana, BB..clarridgeiae,clarridgeiae, BB..rochalimae,rochalimae, BB..washoensis,washoensis, BB..bovis,bovis,BB..henselaehenselae))

ØØ RodentsRodents ((BB..alsatica,alsatica, BB..grahamii,grahamii, BB..vinsonii,vinsonii, BB.. peromysci,peromysci, BB..talpae,talpae, BB..tribocorum,tribocorum, BB..birtlesii,birtlesii, BB..doshiae,doshiae, BB..tayloriitaylorii))

ØØ CattleCattle ((BB..bovis,bovis,BB..henselaehenselae)),, deersdeers ((BB..bovis,bovis, BB..schoenbuchensisschoenbuchensis)),,horseshorses ((BB..henselaehenselae))

ØØ OtherOther:: raccoons,raccoons,wildwildfelids,felids,dolphins,dolphins, monkeys,monkeys, bats,bats,seaseaottersotters

U R

2

20 Fournier - Bartonella

A complex ecology

ØØ Vectors ==Vectors ØØ ((B. henselae++, B. koehlerae, B. clarridgeiae, B. doshiae, B. taylorii, B. quintana))

ØØ Lice ((B. quintana)) ØØ Sandflies ((B. bacilliformis))

ØØ Biting flies ((B. schonbuchensis, B. bovis, B. henselae))

ØØ Ticks ((B. henselae, B. quintana, B. washoensis, B. vinsonii))

U R

Pathophysiology

ØØ IntraIntra--erythrocyticerythrocytic parasitismparasitism=>=>persistantpersistant bacteremiabacteremia ØØ Proliferation ofofmicrovascular endothelialendothelialcellscellsandand neovascularization (angiogenesis)

ØØ ActivationActivationofofthethealternativealternativecomplement pathwaypathway ØØ ReductionReduction ofofthetheoxydativeoxydative responseresponseofofPMNsPMNs (Leboit et al. Am J Surg Pathol. 1989;10:5951989;10:595--610;610; Jacomo et al. Clin Diagn Lab Immunol. 2002;9:8--18)18)2002;9:8

ØØ Manifestations willwillvaryvaryaccordingaccording totothethehosthoststatusstatus (immunocompromised, valvularvalvulardefects)defects) andandthethespeciesspecies

ØØ 1212pathogenicpathogenic speciesspecies:: BB.. henselae,henselae, BB.. quintana,quintana,BB.. bacilliformis, BB..elizabethae,elizabethae, BB..clarridgeiae,clarridgeiae, BB..grahamii,grahamii, BB..vinsonii,vinsonii, BB..koehlerae,koehlerae, BB..alsatica,alsatica, BB..tamiae,tamiae, BB..washoensis,washoensis, BB..rochalimaerochalimae U R

BiphasicBiphasicinfectionsinfections

B. bacilliformis B. quintana B. henselae

Lutzomia verrucarum Pediculus humanus Cat, corporis Ctenocephalides felis,

Inhabitants of Andean Homeless Immunocompetent areas 500-3,000 m patients above sea level Immunocompromised patients Acute infection Oroya fever Trench fever Cat scratch disease

(Gouriet al. (Alexander. BMC Infect Dis. 2007;7:30) Am J Trop Med Hyg. 1995;52:3541995;52:354--9)9)

Verruga peruana Chronic infection EndocarditisBacillary Peliosis hepatis Chronicangiomatosis bacteremia U R

3

21 Fournier - Bartonella

Carrion’s disease

(Alexander. Am J trop Med hyg. 1995;52:3541995;52:354--9)9)

ØØ Or bartonellosis ØØ ØØ Restricted to the Andes mountains ØØ Lutzomia verrucarum ØØ Oroya fever: acute hemolytic anemia, 35% superinfection, 40% mortality

ØØ Verruga peruana (Peruvian warts)

U R

Cat scratch disease

(Debre et al. Soc Med Trop Paris. 1950;66:761950;66:76--99 Wear et al. Science. 1983;221:14031983;221:1403--5)5)

ØØ B. henselae +++, B. clarridgeiae ØØ KittensKittens, Ctenocephalides felis ØØ The most frequent Bartonella infectioninfection ~ 24,000 cases yearly in USA ØØ (Jackson et al. Am J Public Health. 1993:83:17071993:83:1707--11)11)

ØØ 80% <<18 years, worldwide (?) ØØ Mild disease (Carithers. Am J Dis Child. 1985;139:11241985;139:1124--33)33) ØØ Primary inoculation lesion ØØ Regional lymphadenopathy (90% of cases) ØØ Axilla > neck > groin

Self limited, 2 ––6 months6 months (Margileth. Adv Pediatr Infect Dis. 1993;8:11993;8:1--21)21) ØØ U R

Cat scratch disease

ØØ 5050%%feverfever << 3939°°C,C,malaise,malaise, weaknessweakness ØØ 1010%%::locallocalsuppurationsuppuration =>=>needleneedle aspirationaspiration

ØØ RareRare formsforms::bacteremia, hepatichepatic ororsplenicsplenic ,abscess, osteomyelitis, pneumopathy, erythemaerythema nodosum,nodosum, meningomeningo--encephalitis,encephalitis, Parinaud’sParinaud’ssyndromesyndrome (unilateral(unilateral conjunctivitis ++prepre--auricularauricular adenitis),adenitis), Leber’sLeber’sneuroretinitisneuroretinitis (stellate(stellatemacularmacular

exudates)exudates) (Reed et al. Ophtalmology. 1998;105:4591998;105:459--6666 Suhler et al. Ophtalmology. 2000:107:8712000:107:871--6)6)

ØØ Neurological complications ~~11--22%% (Carithers. Am J Dis Child. 1985;139:11241985;139:1124--3333 ofofCSDCSDcasescases Carithers & margileth. Am J Dis Child. 1991;145:981991;145:98--101)101) U R

4

22 Fournier - Bartonella

Trench fever

(Byam et al. Oxford University Press. 1919)

ØØ Or Volhynia fever, Meuse fever, HisHis--WernerWerner disease, shinbone fever, shank fever, quintan fever ØØ B. quintana ØØ Pediculus humanus corporis ØØ WWIWWI ØØ Incubation : 15 --25 days25 days ØØ May be asymptomatic ØØ Most cases: fever with a brutal onset, headache and invalidating muscle pain, sweats, and conjunctive injection ØØ 44--55 days episodes, separated by 4--54 5 days intervals ØØ 33--88 relapses, attenuation of symptoms during recurrences ØØ Rarely, passage to a chronic disease, with weight loss, cardiac arrythmia, dyspnea, anemia, and depression U R

The return of trench fever

(Spach et al. N Engl j Med. 1995;332:4241995;332:424--88 Stein & Raoult. Lancet. 1995;345:4501995;345:450--11 Brouqui et al. N Engl J Med. 1999;340:1841999;340:184--9)9)

ØØ Industrialized countries: homeless people ØØ Infested with body lice ØØ Fever and weight loss ØØ Persistant bacteremia for months without treatmenttreatment 5.3% of homeless in Marseille ØØ (Brouqui et al. Medicine. 2005;84:612005;84:61--8)8)

ØØ Outbreaks in war refugees ((RaoultRaoult et al. LancetLancet. 1998;352:3531998;352:353--8)8)

U R

Bartonella endocarditis

Microorganism MarseillePresent study France 2005 France Great Britain Algeria current study (n(n = = 718) 676) (n = 348) (n = 88) (n = 63) (n = 62)

Medicine Clin Infect Dis Heart 2003; Emerg Infect

2005; 1995; 89:258-62 Dis 2005;

84:162-73 20:501-6 11:216-24

C.Bartonella burnetii sp. 12.733.9 28.448 7.90 12.79.5 22.63.2 Bartonella sp. 12.7 28.4 0 9.5 22.6 Brucella melitensis 0 0 0 0 1.6 sp. 4.6 0 1.1 6.3 3.2 Chlamydia sp. 0 0 2.2 0 0 Staphylococcus sp. 1.9 0 3.4 11.1 6.4 sp. 0.6 0 1.1 0 1.6 T. whipplei 1.8 0.3 0 0 0

CorynebacteriumC. burnetii sp. 33.9 0.6 480 1.1 7.9 12.70 1.6 3.2

Enterobacteriaceae 0.6 0 0 0 00

HACEK bacteria bacteria 0.4 0 0 0 3.2

BrucellaStaphylococcus melitensis sp. 1.90 0 3.40 11.10 1.6 6.4

ChlamydiaStreptococcus sp. sp. 4.60 0 2.2 1.1 6.30 3.20 Other bacteria 3.1 1.1 1.1 1.6 1.6 T. whipplei 1.8 0.3 0 0 0 Fungi 1.2 0 0 6.3 1.6 Other bacteria 3.1 1.1 1.1 1.6 1.6 No aetiology 36.5 22.1 82.9 50.8 54.8 Fungi 1.2 0 0 6.3 1.6 U No aetiology 36.5 22.1 82.9 50.8 54.8 R

5

23 Fournier - Bartonella

Bartonella endocarditis

nd ØØ ~3% of I.E., 2 cause of blood culture-negative endocarditis

ØØ NorthNorth--SouthSouth prevalence gradient

Brouqui and Raoult. FEMS Immunol. Med. Microbiol. 2006;47:12006;47:1--1313

U R

Bartonella endocarditis

ØØ B. quintana++, B. henselae +, B. vinsonii, B. elizabethae, B. koehlerae, B. alsatica

ØØ Males > females ØØ aortic valve > mitral valve ØØ No specific clinical presentation ØØ extensive valvular damage B. henselae B. quintana mean age 47 45 sex ratio M/F 1.5 5.3 previous 90 % 39,5 % valvulopathy (Fournier PE al. Medicine 2001; 80:24580:245--5151 homeless 0 57,8 % Raoult et al. Arch. Intern. Med. 2003;163:2262003;163:226--30)30) body lice 0 36,1 % alcoholism 0 60,5 % contact with 70 % 0 Valvular surgery 90 % 97 %

Death 10 % 8 % U R

Bacillary angiomatosis

Stoler et al. Am J Clin Pathol. 1983;80:7141983;80:714--88 Koehler et al. Clin Infect Dis. 1993;17:6121993;17:612--2424

ØØ BB..henselaehenselae andand BB..quintanaquintana MohleMohle--BoetaniBoetani et al. Clin Infect Dis. 1996;22:7941996;22:794--800800 ØØ Immunocompromised patientspatients (AIDS(AIDS++)++) ØØ InitialInitial symptomssymptoms:: 5050%%cutaneouscutaneous lesions,lesions,2020%% abdominalabdominal pain,pain, 2020%%adenopathyadenopathy andandfeverfever..

ØØ UniqueUnique orormultiplemultiple papules,papules, mostmostoftenoftenred,red, sometimessometimesulcerated,ulcerated, bleeding,bleeding, frequentfrequentsatellitesatellite adenopathies ØØ Progressive increaseincrease ofoflesionlesionsizesize

U R

6

24 Fournier - Bartonella

Bacillary angiomatosis

ØØ Oral, respiratory, anal, and gastrogastro--intestinalintestinal mucosa involvement

ØØ Visceral involvement : liver, spleen, lymph nodes ((B. henselae), bone (), (B. quintana), brain, heart, muscle,

bone marrow Koehler et al. Clin Infect Dis. 1993;17:6121993;17:612--2424

ØØ Without treatment, the evolution is most often fatal

ØØ Few cases of spontaneous cure

ØØ With antibiotics: quasiquasi--constantconstant cure

ØØ Differential diagnosis : Kaposi sarcoma, angioma, bacterial granuloma, cutaneous cancers U R

Peliosis hepatis

ØØ B. henselae ØØ Immunocompromised ØØ Initially described as a visceral manifestation of bacillary angiomatosis ØØ Vascular proliferation => cystic bloodblood--filledfilled spaces in the liver and/or spleen ØØ Fever, abdominal pain, nausea, and diarrhea are inconstant ØØ Frequent hepatomegaly +/+/--splenomegalysplenomegaly ØØ Disseminated hypodense hepatic lesions are observed using CT

U R

Other manifestations

ØØ BB.. henselaehenselae:: prolongedprolongedfever,fever, chronicchronic bacteremiabacteremia

(Slater(Slater etetalal.. NNEnglEnglJJMedMed..19901990;;323323::15871587--9393)),, encephalitis withwithdementiadementia inin HIV+,HIV+,maculomaculo--papulouspapulous eruption,eruption,septicsepticshock,shock, thyroïditis,thyroïditis, urethritis,urethritis, mammarymammarytumor,tumor,

lymphoedema (Lamas(Lamasetetalal..MemMemInstInst OswaldoOswaldoCruzCruz..20082008;;103103::221221--3535))

BB.. quintanaquintana:: chronicchroniclymphadenopathy ØØ (Raoult(Raoult etetalal.. LancetLancet..

19941994;;343343::977977)),,pericarditispericarditis ((JJClinClinMicrobiolMicrobiol..20032003;;4141::52915291--33))

ØØ BB..grahamiigrahamii::uveitisuveitis

ØØ BB..washoensiswashoensis::myocarditismyocarditis

ØØ BB..rochalimaerochalimae::bacteremiabacteremia

U ØØ BB..tamiaetamiae::isolatedisolatedfeverfever R

7

25 Fournier - Bartonella

Diagnosis of Bartonella infectionsinfections SerologySerology

ØØ Cat scratch disease 1:50 << IgG << 1:4001:400 ØØ The sensitivity varies depending on the method: 50 ––88%88% in CSD

ØØ Chronic bacteremia IgG << 1:4001:400

ØØ Endocarditis IgG >> 1:8001:800 ØØ PPV : 97% in endocarditis (Fournier et al. Clin. Diagn. Lab. Immunol. 2002;9:7952002;9:795--801)801)

U R

CrossCross--reactionsreactions

ØØ CrossCross--reactionsreactions between Bartonella, Chlamydia, and CoxiellaCoxiella burnetiiburnetii

(Maurin et al. J Clin microbiol. 1997;35:22831997;35:2283--77 La Scola et al. J Clin Microbiol. 1996;34:22701996;34:2270--4)4)

ØØ Western blot with crosscross--adsorptionadsorption (Houpikian and Raoult. Clin Diagn Lab Immunol. 2003:10:952003:10:95--102)102)

Non adsorbed Adsorbed Bq Adsorbed Bh 1234 1234 1234

1. B. quintana «Fuller» 3. B. vinsonii subsp. berkhoffii 2. B. henselae «Houston» 4. B. elizabethae U R

CultureCulture

ØØ Samples : Heparinized blood, skin, lymph node, cardiac valve or organ biopsies, ......

ØØ Culture on blood agar 5% blood, 5% CO22

ØØ Improved by cell lysis or freezing of clinical specimens

La Scola and Raoult. J. Clin. Microbiol. 1999;37:18991999;37:1899--905905

ØØ Temperature : 3737°°CC except for B. bacilliformis (28(28°°C)C)

ØØ Fastidious bacteria =>primo=>primo-- culture : 1010--45 days45 days U R

8

26 Fournier - Bartonella

Cell culture

ØØ Cell culture (endothelial cells)

ØØ Microculture using the shell vial method

ØØ Inoculation by centrifugation

ØØ Six weeksSix weeks

ØØ Revelation by Gimenez staining, indirect , and/or PCR

U R

Molecular detection

ØØ Samples: arthropods, EDTA blood, skin biopsies, hepatic biopsy, cardiac valve and lymph nodes…

ØØ Genes :Genes : ØØ 1616--23S ITS23S ITS ØØ citrate synthase ØØ groELgroEL ØØ PAP31PAP31 Fournier PE al. Medicine 2001; 80:24580:245--5151 Walls et al. J. Paediatr. Child Health. 2006;42:4692006;42:469--7171 ØØ Identification of Bartonella strainsstrains ØØ Sequencing or speciesspecies--specificspecific U RTRT--PCR assaysassaysPCR R

Mass spectrometric identification

ØØ MALDIMALDI--TOF MSMSTOF ØØ Fast, costFast, cost--effective,effective, reproducible ØØ Enables discrimination of all species ØØ Spectra available in the URMS database (Fournier et al. 2009; unpublished) B. henselae B. quintana

U R

9

27 Fournier - Bartonella

HistologyHistology

ØØ Lymph node, hepatic, cutaneous, cardiac valve biopsies… ..biopsies…

ØØ Silver staining (Warthin(Warthin--Starry)Starry) ØØ Immunohistochemistry (mono(mono--oror polyclonal antibodies)

ØØ Fresh or fixed fragments

ØØ Cat scratch disease: nodularnodular abscessedabscessedlymphadenitis

U R

HistologyHistology

ØØ Bacillary angiomatosis:: Endothelial cell proliferation

U R

HistologyHistology

ØØHepatic peliosis: Dilatation of hepatic sinusoidssinusoids

U R

10

28 Fournier - Bartonella

HistologyHistology

ØØ Endocarditis: Small vegetations Severe fibrosis HPSHPS Lepidi H et al. Am. J. Clin. Pathol. 2000;114:8802000;114:880--99

HPSHPS

U WarthinWarthin--StarryStarry IHCIHC R

Treatment of Bartonella infectionsinfections

Penicillin G / Amoxicilline : 0.06 µg/mL Cefotaxime : 0.25 µg/mL Imipeneme : 0.5 µg/mL0.5 µg/mL Gentamicin : 1 µg/mL1 µg/mL Doxycycline : 0.12 µg/mL Rifampicine : 0.25 µg/mL Erythromycine : 0.12 µg/mL Clarithromycine : 0.015 µg/mL Sparfloxacine : 0.12 µg/mL Sulfamethoxazole : 5 µg/mL

ØØ MICs correlate poorly with in vivo efficacy ØØ Only aminoglycosides are bactericidal (Musso et al. J Antimicrob Chemother. 1995;36:1011995;36:101--88 Rolain et al. J. Antimicron Chemother. 2000;46:8112000;46:811--4)4) U R

Treatment

ØØ No single treatment for all Bartonella infectionsinfections

ØØ Cat scratch disease without No antibiotic, or azithromycin, 500 mg, 1st day,

then 250 mg/d, 4 days Bass JW et al. Pediatr Infect Dis J. 1998;17:447-52

ØØ Neurological involvement, retinitis Doxycycline 200 and rifampin 600 mg/d, 6 weeks

ØØ Trench fever Gentamicin 3 mg/kg/d, 2 weeks and doxycycline 200 mg/d, 4 weeks U R

11

29 Fournier - Bartonella

Treatment

ØØ Bacillary angiomatosis, peliosis hepatis Double effect: antianti--microbialmicrobial and anti--angiogenicanti angiogenic

(Meghari et al. J Infect Dis. 2006;193:3802006;193:380--6)6) Erythromycin 500 mg x 4/d, 3 months (BA), 4 months (PH)

ØØ Endocarditis, bacteremia gentamicin 3mg/kg/d, 2 to 3 weeks + doxycycline 200 mg/d or amoxicillin 200mg/kg/d, 6 weeks6 weeks

U R

Conclusions

ØØ BartonellaBartonella sp. are emerging and reemerging pathogenspathogens

ØØ Zoonotic agents

ØØ 12 human pathogens, more to discover?

ØØ A wide array of clinical manifestations

ØØ Mild (CSD) to severe (Oroya fever, BA, endocarditis)

ØØ Specific antibiotic regimens U R

Case 1

Mr P, 39 years-old, a French legionnaire and daredevil, was admitted to hospital on April 15, 2009, for persistent dyspnea that had started 4 months earlier in Afghanistan, and had progressively worsened. The patient had a medical history of rheumatic aortic valvulopathy that required a valve replacement with a bioprosthesis at age 25. In 2001, while serving in Kosovo, he developed a Q fever endocarditis and was treated for 18 months with doxycycline and hydroxycholoroquine. In 2001, his aortic bioprosthesis was changed for an homograft. On admission in April 2009, he had a fever of 39.1°C, a weight loss of 10 kgs, and transoesophageal echocardiography revealed a severe aortic insufficiency with small vegetations. His blood tests showed a white blood cell count of 15.000/mL (90% PMN) and a creatinin level of 200 µmol/mL. Three blood cultures remained sterile. Q fever serology was also negative.

U R

12

30 Fournier - Bartonella

Question 1

Do you think that this patient may have a Q fever relapse?

A. Yes

B. No

Question 2

The valvular insufficiency required a third aortic valve replacement, with a mechanical prosthesis. PCR assays targeting 2 distinct genes identified . What epidemiological risk factor will you search?

A. Contact with dogs

B. Contact with cats

C. Contact with rats

D. None of these

Question 3

What antibiotic therapy do you propose?

A. Hydroxycloroquine + doxycycline

B. Amoxicillin + gentamicin

C. Doxycyclin + rifampin

D. Any of these three

13

31 Fournier - Bartonella

Case 2

Mr B, 65 years-old, homeless and alcoholic (2 liters of whiskey per day), was admitted to hospital for persistent fever. The patient had lived in the streets for 15 years. He had not known history of valvulopathy. On admission, he had a fever of 38.7°C and several cutaneous scratching lesions. Trans-thoracic ultrasound showed aortic vegetations of 14mm and a severe aortic insufficiency. His blood tests showed a white blood cell count of 9.100/mL (80% PMN), a C reactive protein level of 101 and an ESR of 45mm, 1st hour. Three blood cultures remained sterile. Q fever serology was negative but the patient exhibited an IgG titer to Bartonella sp. of 1:800.

Question 1

What diagnosis do you suspect?

A. B. quintana endocarditis

B. B. henselae endocarditis

C. B. alsatica endocarditis

D. B. bacilliformis endocarditis

Question 2

Would an IgG titer to Bartonella sp. lower than 1:800 rule out the diagnosis of Bartonella endocarditis?

A. Yes

B. No

14

32 Fournier - Bartonella

Question 3

In this case, what complementary test could you perform?

A. Prolonged blood culture

B. Specific PCR from EDTA blood

C. Western blot +/- cross-adsorption

D. All of these tests

!! Attention !! Haemobartonella BartonellaBartonella ØØ ≠≠ ØØ Haemobartonella andand Eperythrozoon werewere reclassifiedreclassified amongamong

(Neimark(Neimarketetalal..IntInt JJSystSystEvolEvolMicrobiolMicrobiol..20012001;;5151::891891--99))

ØØ ButBut thethe confusionconfusion stillstill existsexists

ØØ BB..murismuris == HH..murismuris

U R

15

33 34 Gikas –Chronic Q fever

WHO Collaborating Center for Research and Training in Mediterranean Zoonoses A.Gikas Assoc Prof. Infectious Diseases Chronic Q fever: from diagnosis to treatment

UNIVERSITY OF CRETE - Faculty of Medicine Laboratory of Clinical Bacteriology Parasitology, Zoonoses and Geographical Medicine

Coxiella burnetii Ενδοκαρδίτιδα από : περιγραφή ενδιαφέροντος AA χχχχ περιστατικού Αλεξάνδρα Τζουμάνη11, Αθηνά Λιονή11, Μαρία Χίνη11, Γεώργιος Μηλάτος11, Κω νσταντίνος Λιάγκας22, Αχιλλέας Γκίκας33, Γεώργιος Οικονομόπουλος44, Μάριος Κ. Λαζανάς11

1 Γ΄ Παθολογικό Τμήμα-Μονάδα Λοιμώξεων Γ.Ν.Α. «Κοργιαλένειο –Μπενάκειο» Ε.Ε.Σ. 2 Β΄ Καρδιολογικό Τμήμα, Γ.Ν.Α. «Κοργιαλ ένειο –Μπενάκειο» Ε.Ε.Σ. 3 Εργαστήριο Κλινικής Βακτηριολογίας-Παρασιτολογίας, Ζωονόσων και Γεωγραφικής Ιατρικής, Πε.Πα.Γ.Ν.Ηρακλείου 4 Β΄ Καρδιοχειρουργικό Τμήμα, Γ.Ν.Α. «Ιπποκράτειο» ΕΙΣΑΓΩΓΗ-ΣΚΟΠΟΣ ΠΑΡΟΥΣΙΑΣΗ ΠΕΡΙΣΤΑΤΙΚΟΥ Η ενδοκαρδίτιδα αποτελεί την κυριότερη κλινική εκδήλωση χρόνιου Άνδρας 71 ετών, εισήχθη στην Κλινική μας για διερεύνηση παρατεινομένου πυρετού Q. Η ενδοκαρδίτιδα από C. burnetii αντιπροσωπεύει το 3-5% εμπυρέτου. 10 του συνόλου των ενδοκαρδιτίδων και περίπου το 50% των Η παρούσα ν όσος χρονολογείται από εβδομάδων, με καταβολή, εύκολη κόπωση . ενδοκαρδιτίδων με αρνητικές αιμοκαλ λιέργειες. και αν ορεξία Παρουσιάζουμε μια σ πάνια περίπτωση ενδοκαρδίτιδας από C. burnetii Δύο μήνες πριν την εισαγωγή του ν οσηλεύτηκε σε άλλο νοσοκομείο λόγω δύσπνοιας, βήχα και πυρετού. Αντιμετωπίσθηκε ως λοίμωξη αν απνευστικού με σε ασθενή με στένωση αορτικής βαλβίδας. μοξιφλοξασίνη και αμοξυκιλλίνη/κλαβουλανικό και τοποθετήθηκε βηματοδότης λόγω πλήρους κολποκοιλιακού αποκλεισμού, εναλλαγής RBBB/LBBB και βραδυκαρδίας. ΣΥΜΠΕΡΑΣΜΑΤΑ Δύο εβδομάδες προ της εισαγωγής του, εισήχθη σε Καρδιολογική Κλινική για • επανεκτίμηση της στέν ωσης της αορτικής βαλβίδας όπου διαπιστώθηκε πυρετός έως Σε κάθε περίπτωση ενδοκαρδίτιδας με αρνητικές αιμοκαλ λιέργειες θα 38 C, . C. burneti i. ο διαλείπων , χωρίς ρίγος, καλά αν εκτός και χωρίς ιδιαίτερα συν οδά συμπτώματα πρέπει να γίνεται έλεγ χος για . • Ο ασθεν ής μεταφέρθηκε στην Κλιν ική μας για περαιτέρω διερεύν ηση Συχνά, η τεκμηρίωση της διάγνωσης καθυστερεί λόγω ατύπων Από την αντικειμενική εξέταση διαπιστώθηκε πληκτροδακτυλία, πλήρης αρρυθμία κλινικών συμπτωμάτων, μη διαγνωστικών υπερηχογραφ ημάτων 4/6 . 12%) . και συστολικό φύσημα στην εστία ακρόασης της αορτικής βαλβίδας Από τον (παρουσία εκβλαστήσεων στο και αρνητικών αιμοκαλ λιεργ ειών : AMK Ra-test 281 IU/ml, IgG • εργαστηριακό έλεγχο στείρες, μονοκλωνική κ, Θεραπεία εκλογ ής αποτελεί ο συνδυασμός δοξυκυκλίνης και κρυοσφαιρίνες θετικές. Διαθωρακικό και διοισοφάγειο U/S καρδίας: Διάταση αορτικής υδροξυχλωροκί νης για χρονικό διάστημα ≥ 18 μηνών, ενώ η θνητότητα 3-10%. ρίζας και ανιούσης αορτής, ασβέστωση γλωχίν ων αορτικής βαλβίδας με σοβαρού κυμαίνεται από βαθμού στέν ωση και μικρή διαφυγή, απουσία εκβλαστήσεων, ΕF: 58%. Αντισώματα • Απαιτείται συχνή κλινική και ορολ ογική παρακολούθηση για εκτίμηση έναντι C.burnetii (IFA): IgG Phase I 1/32768, IgM Phase I 1/512, IgG Phase II 1/16384, της ανταπόκρισης στη θεραπεία και έγκαιρη διάγνωση των IgM Phase II 1/512 . . υποτροπών Στον ασθενή χορηγήθηκε δοξυκυκλίνη και σιπροφλοξασίνη. Πέντε ημέρες μετά, παραπέμφθηκε σε Kαρδιοχειρουργική Κλινική για επείγουσα χειρουργική ΒΙΒΛΙΟΓΡΑΦΙΑ αν τιμετώπιση λόγω περαιτέρω επιδείνωσης της καρδιακής λειτουργίας (EF:36%). Τα 1. Hartzell JD, Wood-Morris RN, Martinez LJ, Trotta RF. Q fever: epidemiology, διεγχειρητικά ευρήματα ήταν: διάταση αορτικής ρίζας (6 cm), ευρέως ασβεστωμένη diagnosis , and treatment. Mayo Clin Proc. 200 8 May; 83(5):574 -9 αορτική βαλβίδα με διάχυτες διηθήσεις από ράκη (εκβλαστήσεις) και υποβαλβιδικό 2. Houpikian P, Raoult D. Blood Culture–Negative Endocarditis in a Reference απόστημα στο μεσοκοιλιακό διάφραγμα (2Χ4 cm). Center. Etiologic Diagnosis of 348 Cases. Medicine (Baltimore) 2005 May; PCR στο χειρουργικό παρασκεύασμα της βαλβίδας: θετικό για C. burnetii. 84(3):162 -73 Η μετεγχειρητική πορεία υπήρξε ομαλή και ο ασθενής εξήλθε από την Κλινική μας με 3. Raoult D, Houpikian P, Tissot Dupont H, et al. Treatment of Q fever αγωγή: δοξυκυκλίν η 100 mg Χ 2 και υδροξυχλωροκίν η 200 mg Χ 3 po. endocarditis: Comparison of 2 regimens containing doxycycline and ofloxacin or hydroxychloroquine. Arch Intern Med. 199 9 Jan 25;159 (2):167 -73 4. Tselentis Y, Gikas A, Kofteridis D, Kyriakakis E, Lydatakis N, Bouros D. Tsaparas N. Q fever in the Greek isla nd of Crete: epidemiologic, clinical, and therapeutic data from 98 cas es. Clin Infect Dis. 199 5 May;20(5):131 1-6

Η παραπάνω Αναρτημένη Ανακοίνωση, παρουσιάσθηκε κατά την διάρκεια του 9ου Πανελληνίου Συνεδρίου Λοιμώξεων, Αθήνα 6-8/02/2009, με αύξοντα αριθμό ΑΑ χχχ

The Case (1)

q 71-y.o./M, Hospitalized for investigation: 10 weeks fever up to 380C, accompanied by malaise, fatigue, and anorexia q History of aortic valve stenosis, q 8 weeks before: Admission in another hospital for respiratory infection which was complicated by AV block and arrhythmia q Treated with moxifloxacin & amoxicillin/clavulanate, and placement of a pacemaker 0 q Six weeks later, follow-up: Fever up to 38 C well tolerated è admitted for further evaluation. q O/E: arrhythmia, systolic aortic valve murmur, clubbing q Laboratory results: negative blood cultures, Ra-test 281 IU/ml, positive testing for monoclonal IgG and cryoglobulins. q Transthoracic and transesophageal echocardiography showed aortic valve stenosis and calcification, an ejection fraction of 58%, but no vegetations.

35 Gikas –Chronic Q fever

C.burnetti or IG?

D. Raoult et al J.Clin.Microbiol, 2005, p. 5238–5242

The Case 2

Antibodies for C.burnetii were found as follows:

anti-phase I IgG 1:32768,

anti-phase I IgM 1:512,

anti-phase II IgG 1:16384,

anti-phase II IgM 1:512.

The Case 3

q The patient was treated with doxycycline and ciprofloxacine,

q 5 days later deterioration of his heart function (EF 36%) necessitated the performance of emergency cardiosurgery,

q With the following observations: dilatation of the aortic root (6 cm), aortic valve calcification, stenosis, vegetations, an abscess in the interventricular septum.

q Valve tissue PCR was positive for C.burnetii.

36 Gikas –Chronic Q fever

Q FEVER

q Caused by Coxiella burnetii, an obligate intracellular bacterium q Wide spectrum of clinical manifestations

•Q fever can be 1. Acute 2. Chronic, 3. Long-term sequelae are gaining acceptance as a third category of the disease.

q More than 30 different clinical syndromes have been described

From Acute to Chronic Q fever

Raoult D, Parola P, eds. Rickettsial Diseases. Informa healthcare 2008.

37 Gikas –Chronic Q fever

Q FEVER-The acute disease

q The classic presentation is: A flulike illness manifested by Fevers, sweats, cough, myalgias, and arthralgias.

q 50%-60% patients who are infected with Q fever are asymptomatic

q Three main presentations predominates: Isolated fever, hepatitis, or

•Other presentations: 1. Neurologic signs occur in 1% 2. Cardiac involvement is found in 2% 3. Long-term cardiovascular complications

Evolution to chronicity

Risk of evolution to chronic disease

a. Patients with -Valvular abnormalities 38% will develop endocarditis within 2 years -Aneurysms or vascular grafts

b. Pregnant women

c. Immune suppression (HIV, cancer, lymphoma, splenectomy, treatment)

d. The evolution of acute Q fever to chronic fatigue syndrome has been described

CHRONIC DISEASE Endocarditis The most common form of chronic Q fever (60%-70%),

3% to 5% of all endocarditis cases

1/1.000.000 habitants

Estimated to occur in 0.76% of acute Q fever cases >800 cases are published 264 cases in France 227 cases U.K, Ireland 62 in Spain cases, 35 cases in Israel 21 cases in Switzerland 18 cases in Australia 10 in Canada cases Parker NR, et al. Lancet; 2006. Fenollar F, et al. Clin Infect Dis; 2001. 5 cases in Greece

38 Gikas –Chronic Q fever

Endocarditis The clinical presentation

There is an average of 12 months between onset and diagnosis

Manifestations range from asymptomatic to congestive heart failure

Fever absent in up to 18% of patients

These pts are often misdiagnosed

Marrie TJ, Raoult D. Int J Antimicrob Agents; 1997.

The clinical presentation

Raoult D, Parola P, eds. Rickettsial Diseases. Informa healthcare 2008.

Q fever Endocarditis-Diagnosis I

Suspicion: Valvular abnormalities,

Presenting with evocative clinical signs fever, hepatitis, tiredness, clubbed fingers, weight loss, renal failure, purpuric rash

Laboratory findings :↑ESR, y liver enzymes, thrombocytopenia, glomerulonephritis

Serology in Modified Duke’s criteria

39 Gikas –Chronic Q fever

Serology Reference technique: Indirect immunofluorescence

Acute Q fever: antibodies against phase II antigens (2-3 w) Chronic Q fever: characterized by the presence of anti-phase I antibodies An IgG anti-phase I antibody titer of >800 is considered diagnostic of Q fever endocarditis

Dupont, H. T., et al. Clin Diagn Lab Immunol; 1994. Raoult, D., et al. Medicine (Baltimore);2000.

Raoult D, Parola P, eds. Rickettsial Diseases. Informa healthcare 2008.

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6

Age / Sex 41 / M 53 / M 58 / M 35 / M 56 / M 71 / M Underlying Congenital AoV None AoV stenosis & abscess 5 AoV stenosis Surgery for AoV AoV stenosis, conditions –Medical stenosis operated months before treated with stenosis 2 months LRTI treated with History 27 y ago, Mitral Surgery & Antibiotics before Antibiotics & Pacemaker 2 valve prolapse months before

Duration of 3 months 3 weeks 3.5 months 5 months 2 months 2 weeks symptoms Fever Yes 390C Yes, 39-400C 38-390C 380C

Other signs and Rigor, fatigue, Rigors, malaise, Rigor Rigors, sweating, Malaise, fatigue, anorexia, symptoms malaise fatigue, myalgia, intense malaise, clubbing breath shortness weight loss, myalgia Auscultation findings AoV Systolic and AoV Changing Arrhythmia, AoV diastolic murmur heart murmur Systolic murmur TTE / TEE findings None New Aortic valve None Aortic valve stenosis,

abscess on aortic valve vegetation calcification, ΕF 58% Serology Anti-phase I IgG Anti-phase I IgG Anti-phase I IgG Anti-phase I IgG Anti-phase I IgG Anti-I IgG 1:32768, 1:30720 1 1:1024 1:1024 1:15360 1:3200 Anti-I IgM 1:512, Anti-II IgG 1:16384, Other tests performed PCR on valve Blood PCR (+) PCR on valve tissue (+) tissue (+) (+) Surgical findings Treatment Rifampicin Aortic valve Doxycycline & Quinolone Empirical, Empirical, Switched Doxy & Quinolone, & Doxycycline replacement, Switched to to doxy & Surgery & Switched to Doxy & Doxy upon hydrochloquine Doxy & hydrochloquine Quinolone diagnosis Favorable Outcome Yes Yes Yes Yes Yes Yes Kokkini S,et al. Clin Microbiol Infect; 2009 (In Press)

40 Gikas –Chronic Q fever

The prognosis of chronic Q fever

The mortality rate Was 37% in a series of 76 patients

reported in 1987, Raoult D, et al. J Infect Dis; 1987. Was only 15% in more recent series of 116 patients, between 1997 and 2000, under 5% among the most recently diagnosed patients Raoult D, et al. Arch Intern Med; 1999. Earlier diagnosis, an efficient long-term treatment, and a better follow-up

Siegman-Igra Y,et al.Scand J Infect Dis;1997.

Chronic Q Fever-Vascular infection (aneurysms or vascular grafts) The second most frequent presentation >45 cases reported in France, U.K., U.S.A., Switzerland, and Australia

Fournier PE, et al. Clin Infect Dis; 1998. Mejia A. Ann Vasc Surg; 2000. Botelho-Nevers E, et al. Eur J Clin Microbiol Infect Dis; 2007.

Aortic aneurism -infected -intestinal fistula or a spondylitis, Vascular graft

Senn L, et al. BMC Infectious Diseases; 2005. Sessa C, et al. Vasc Surg; 2005.

Pregnancy and Chronic Q Fever

Infection by C. burnetii during pregnancy Risk of chronic Q fever in the mother Related to the duration of the disease during the pregnancy It is not prevented by treatment during pregnancy One-half of infected show a chronic infection serological profile Repeated abortions or preterm births are observed during later pregnancies

Raoult et al. Arch Intern Med; 2002.

41 Gikas –Chronic Q fever

Immunodeficiency and Chronicity

HIV-infected,lymphomas patients

High risk of relapse or evolution to chronic Q fever

Raoult D,et al. Clin Infect Dis; 1992.

Other manifestations of chronic Q fever

Osteoarticular infections Landais C,et al. Eur J Clin Microbiol Infect Dis; 2007.

Chronic hepatitis in alcohol addicts Raoult D,et al. Medicine (Baltimore); 2000. Pseudotumors of the spleen or the lung

Lipton JH,et al. Chest; 1987. Infection of a ventriculo-peritoneal drain

Lohuis PJ,et al. Neth J Med; 1994. A case of vascularitis with pulmonary amyloidosis Kayser K,et al. Respiration; 1995.

Treatment Endocarditis I

Tetracycline: the mainstay of treatment

Combination therapy due to reported recovery of viable C.burnetii after years of therapy with Doxycycline

→ HCQ 200mgX3 daily & Doxy 100mgX2 daily

(check for G6PD-HCQ and warn for photosensitivity by Doxy)

Raoult, D,et al. Arch Intern Med; 1999. Hartzell JD,et al. Mayo Clin Proc; 2008.

42 Gikas –Chronic Q fever

Treatment Monitoring Doxycycline plasmatic level must remain over 5g/ml Rolain JM,et al. J Infect Dis; 2003. If the strain is cultured from blood or valves z (MIC) of doxycycline to C.burnetii

Plasmatic levels should be adjusted between 1.5 and 2 MICs Rolain JM,et al. Antimicrob Agents Chemother; 2005. Hydroxychloroquine 200x3 three months 200 mg BID after three to six months, and 200 to 300 mg a day after six months.

Hydroxychloroquine plasmatic levels should be above 10.2 mg/L

Treatment Duration

Combination therapy for minimum 18 Stein A,et al. Eur Heart J;1995. months Brouqui P,et al. Arch Intern Med;1993.

Some recommend life-long doxy after initial combination treatment

Maurin M, Raoult D. Clin Microbiol Rev;1999. Treatment until phase I IgG, IgA <1:200

Follow-up IgG antibodies remain positive for years. Raoult D,et al. Arch Intern Med; 1999. A twofold decrease in phase I IgA and phase I IgG is expected after one year of treatment

Treatment monitoring & Serologic testing every month for one year n every three months for two years n every six months thereafter n After three years, treatment can be stopped if phase I IgG < 200 Stein A,et al. Eur Heart J; 1995. Brouqui P,et al. Arch Intern Med; 1993.

43 Gikas –Chronic Q fever

TT - Endocarditis II

Alternative combination regimens used: Trimethoprim-sulfamethoxazole & n Rifampicin Doxycycline & Rifampin n Quinolone & Doxycycline n Trimethoprim-sulfamethoxazole & n Doxycycline Maurin M, Raoult D. Clin Microbiol Rev;1999.

Surgical valve replacement Valvular damage n Heart failure n Raoult, D. Antimicrob Agents Chemother;1993.

Treatment

Other forms of Chronic Q fever - Vascular infections

Surgical treatment associated n with survival

Antibiotics individualized n Doxy & HCQ n

Botelho-Nevers E,et al. Eur J Clin Microbiol Infect Dis;2007.

Treatment

Other forms of Chronic Q fever - - Osteoarticular infections

Extremely rare n

Surgical debridement n + 18mo Doxy & HCQ ± Rif n

Landais C,et al. Eur J Clin Microbiol Infect Dis;2007.

44 Gikas –Chronic Q fever

Treatment

Other forms of Chronic Q fever – Q fever in pregnancy Not recommended antibiotics: Doxy, FQ, HCQ

Cotrimoxazole until delivery No cure n Decreased spontaneous abortion, intrauterine n growth retardation, intrauterine fetal death, premature delivery, oligohydramnion Treat mother after birth with Doxy&HCQ n Doxy and C.burnetii excreted in breast milk n

Carcopino X,et al. Clin Infect Dis; 2007. Raoult D,et al. Arch Intern Med; 2002.

Clinical response Effective antibiotic therapy Afebrile within one week n Hepatomegaly and splenomegaly disappear n within 2-12 weeks. Aminotransferases and thrombocytopenia n slowly return to normal

There is at present no specific test that makes it possible to conclude that a patient has definitively been cured

Levy PY,et al. Eur J Epidemiol;1989.

Prevention of Chronicity I

In people with risk factors, and acute Q fever, symptomatic or not,

Investigation : a. pregnancy test in women, b. cardiac ultrasound c. A cause of immune suppression (HIV, cancer, lymphoma, splenectomy, treatment).

Treatment: As a Chronic case treatment for one year

Fenollar F,et al. Clin Infect Dis;2001.

45 Gikas –Chronic Q fever

Landais C, Fenollar F, Thuny F, Raoult D. Clin Infect Dis; 2007 .

Prevention -

Whole-cell vaccine licensed in Australia (Q- Vax) 1985-90: no Q fever in 2555 vaccinated n persons Vs 55 cases in 1365 unvaccinated Prevaccination screening: serology (-), skin n test (-), Q fever history (-)

Kermode M,et al. Aust N Z J Public Health; 2003. Ackland J,et al. Med J Aust; 1994. Marmion BP,et al. Epidemiol Infect; 1990.

Acellular (trichloroacetic acid, chloroform-methanol residue) with few data available Parker NR,et al. Lancet; 2006.

46 Gikas –Chronic Q fever

Τηλ. Εργαστηρίου Ρικκετσιών 2810-394624 ••[email protected] of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine, WHO Collaborating Center, University Hospital of Heraklion,Tel+30-2810-394624 :, Fax:302810392847 [email protected]

47 48 Summary

Role of emerging intracellular bacteria in pneumonia G. Greub (Lausanne, CH)

Pneumonia is a common disease associated with significant morbidity and mortality. To guide antimicrobial therapy and public health measures, it is important to identify the aetiological agent of pneumonia. However, an agent is only identified in about 50% of cases. Intracellular bacteria, which are not detected using conventional media used in diagnostic laboratory, may be the agents of some of these pneumonia cases of unknown aetiology. Several Chlamydia-related bacteria recently emerged as possible agents of pneumonia. One of them, Parachlamydia acanthamoebae, has been initially identified as a possible human pathogen during the investigation of an outbreak. The role of Parachlamydia in pneumonia is now further supported by numerous clinical studies, by the growth of this Chlamydia-like organism in vitro in human pneumocytes and macrophages, and by animal models of lung infections. Like , Parachlamydia may resist to the microbicidal effectors of free-living amoebae (Figure). This is of importance since free-living amoebae may represent a potent and widespread evolutionary crib for the internalized bacteria, leading to acquired resistance to another phagocytic cell, the human alveolar macrophage. Furthermore, amoebae may be used to selectively grow new bacterial species that may have evolved to resist macrophages and thus became pathogenic. Using amoebae as cell background in an amoebal co-culture system, several new amoebae-resisting bacteria have recently been discovered, including new clades within the Chlamydiales order. The current challenge is to specify the role of these amoebae-resisting microorganisms in lower respiratory tract infections. In the future, it will also be important to define the role of these intracellular bacteria in other clinical settings.

Figure: Presence of large numbers of Parachlamydia acanthamoebae strain BN9 within Acanthamoeba polyphaga, as seen by electron microscopy; 3'500x magnification.

49 50 Greub –Intracellular bacteria in pneumonia

The role of emerging intracellular bacteria in pneumonia

Gilbert Greub, MD PhD Institute of Microbiology and Infectious diseases service University Hospital Center and University of Lausanne Switzerland

Defining the role as an agent of pneumonia of newly identified intracellular bacteria

Why is it important to identify the etiological agent of a pneumonia ?

Why novel intracellular bacteria will likely be identified as new emerging agents of pneumonia ?

How to identify new possible agents of pneumonia and how to precise their pathogenic role ?

Pneumonia Lung infection

Frequent, significant mortality

51 Greub –Intracellular bacteria in pneumonia

Pneumonia Lung infection

Frequent, significant mortality

Crucial to define the etiology - to choose an optimal antibiotic treatment

Pneumonia Lung infection

Frequent, significant mortality

Crucial to define the etiology - to choose an optimal antibiotic treatment - to guide public health measures

Coxiella Legionella Influenza A H1N1

Pneumonia Lung infection

Frequent, significant mortality

Crucial to define the etiology - to choose an optimal antibiotic treatment - to guide public health measures

Unknown etiology in 50% of cases

virus

Mainly discovered during outbreaks

52 Greub –Intracellular bacteria in pneumonia

Etiology of pneumonia unknown

- 50% of community-acquired pneumonia

Bochud et al. Medicine 2001 Falguera et al. Arch Intern Med 2001 - 75% of nosocomial pneumonia Costa et al. Int J Antimicrob Agents 2001

Virus Bacteria (antibiotics)

Mainly discovered during outbreaks

Strict intracellular bacteria Remain undetected with axenic media routinely used in diagnostic laboratory

Multiply only in cells

2

1

Macrophage (ConA) 1 Bacteria (Chlamydiales) 2 Q1

Intracellular bacteria Mainly discovered during outbreaks

Chlamydia psittaci - outbreak in Uster in 1880 psittacosis - pandemia in 1929

53 Greub –Intracellular bacteria in pneumonia

Intracellular bacteria Mainly discovered during outbreaks

Chlamydia psittaci - outbreak in Uster in 1880 psittacosis - pandemia in 1929

Legionella pneumophila Fraser et al. 1977 New Engl J of Med « Legionnaires’disease: description of an epidemic of pneumonia »

Lung Guinea pig Agar

Legionella pneumophila No interhuman transmission Infection by aerosols

Water Free-living amoebae Q2

Free-living amoebae : A reservoir for Legionella spp.

Rowbotham. J Clin Pathol, 1980

Lausanne’s University Hospital

Samples positive Amoebae No amoebae for Legionella 33% 3% (p<0.001)

Thomas et al., Appl Env Microbiol, 2006

54 Greub –Intracellular bacteria in pneumonia

Free-living amoebae: an evolutionary crib

Selection of Virulence traits

Environnement

Adaptation to macrophages Lower respiratory tract

Adapted from: Greub et al. Clin Microb Rev 2004

Other amoebae-resisting bacteria

•may (like Legionella) resist to amoebae

•might also survive to another phagocytic cell: the human macrophage

•are good candidate as agents of pneumonia of unknown etiology

Better not to wait for the next outbreak…

To discover new pathogenic intracellular bacteria

1. To discover new bacterial species

Amoebae as a tool

2. To precise their role as agents of pneumonia Parachlamydia

55 Greub –Intracellular bacteria in pneumonia

To discover new bacterial species

Respiratory tract samples, environment

Amoebal co-culture Amoebal enrichment

New species

Co-culture Culture d’amibes

Incubate at28°C migration

Investigated sample No lysis serial dilutions

Photo Lyse/non lysées migration

Lysis

Co-culture Amoebal enrichment

Incubate at 28°C migration

Investigated sample

serial dilutions

Photo Lyse/non lysées migration

56 Greub –Intracellular bacteria in pneumonia

To discover new bacterial species Respiratory tract samples, environment

Amoebal co-culture Amoebal enrichment

New species

Estrella lausannensis Criblamydia sequanensis

Chlamydiales order

Parac hlamy dia acantham oeb ae Bn9 Y07556 79 Parac hlamy dia st rain Seine 1 00 Parac hlamy dia Halls c occ us AF366 365

64 Endo sy mbiont UV 7 AJ71541 0

7 3 EndosProtochlamydia ym biont UW amoebophila E25 BX908798 UWE25 10 0 ProtochlamydiaKnic 2A naegleriophila strain KNIC 9 0 Endos ym biont U WE1 AF0 836 14 Ne ochlam y dia hartm annellae AF17 7275 « Chlamydia-like » 68 10 0 Endosy mb iont TUM E1 AF 0983 30 10 0 Endosy mbiont UW C22 AF 0836 16

6 3 Waddlia c hon drophil a AF042 496 10 0 W addlia m alay si ens is AY18480 4

4 9 Cribl am y dia s equ anen si s

10 0 Rhab doc hlamydi a crass ificans A Y9280 92 Rha bdo chl am yd ia porcellionis AY22386 2

91 S imk ania nege vensi s U684 60

10 0 Frits chea bem is iae AY140 910 10 0 Frits ch ea erio coc ci AY14091 1 C hlamydi ophila pneum oniae L061 08

8 3 Ch lam ydi a murida rum D85 718 10 0 10 0 Ch lamy dia trac hom atis D8 9067 Ch lamy dia sui s U731 10

48 Chl am y diophila pec orum D8 8317 « Chlamydia » Chl am y diophila felis D85 701 8 7 Chlam yd iophil a c aviae D8 570 8 1 00 79 C hlamydi ophila abortus AB0017 83 86 Chl am y diophila psi ttac i A B00177 8 Pis ci chl am yd ia s almonis AY462 244

0 .02

Chlamydiales order Families

Parac hlamy dia acantham oeb ae Bn9 Y07556 79 Parac hlamy dia st rain Seine 1 00 Parac hlamy dia Halls c occ us AF366 365 64 Endo sy mbiont UV 7 AJ71541 0

7 3 EndosProtochlamydia ym biont UW amoebophila E25 BX908798 UWE25 Parachlamydiaceae 10 0 ProtochlamydiaKnic 2A naegleriophila strain KNIC 9 0 Endos ym biont U WE1 AF0 836 14 Ne ochlam y dia hartm annellae AF17 7275

68 10 0 Endosy mb iont TUM E1 AF 0983 30 10 0 Endosy mbiont UW C22 AF 0836 16 6 3 Waddlia c hon drophil a AF042 496 Waddliaceae 10 0 W addlia m alay si ens is AY18480 4

4 9 Cribl am y dia s equ anen si s Criblamydiaceae 10 0 Rhab doc hlamydi a crass ificans A Y9280 92 Rhabdochlamydiaceae Rha bdo chl am yd ia porcellionis AY22386 2 91 S imk ania nege vensi s U684 60 Simkaniaceae 10 0 Frits chea bem is iae AY140 910 10 0 Frits ch ea erio coc ci AY14091 1 C hlamydi ophila pneum oniae L061 08

8 3 Ch lam ydi a murida rum D85 718 10 0 10 0 Ch lamy dia trac hom atis D8 9067 Ch lamy dia sui s U731 10

48 Chl am y diophila pec orum D8 8317 Chlamydiaceae Chl am y diophila felis D85 701 8 7 Chlam yd iophil a c aviae D8 570 8 1 00 79 C hlamydi ophila abortus AB0017 83 86 Chl am y diophila psi ttac i A B00177 8 Pis ci chl am yd ia s almonis AY462 244

0 .02

57 Greub –Intracellular bacteria in pneumonia

To discover new pathogenic intracellular bacteria

1. To discover new bacterial species

Amoebae as a tool

2. To precise their role as agents of pneumonia Parachlamydia

Q3

Parachlamydia acanthamoebae within Acanthamoeba castellanii

a-Pa, a-Ig+Alexa488 ConA+Alexa594 DAPI Greub G, Clin Microbiol Infect, 2009.

Parachlamydia acanthamoebae

Greub G. et al. Appl Env Microbiol 2002; 68:3076-3084 Greub –Intracellular bacteria in pneumonia

Parachlamydia acanthamoebae

Greub et al. Clin Microb Review 2004

Parachlamydia and pneumonia

Epidemiological hint - Isolation of an amoeba containing Parachlamydia from the water of an humidifier associated to an outbreak of fever

Birtles et al. Lancet 1997

Greub G.

Parachlamydia and pneumonia

Epidemiological hint - Isolation of an amoeba containing Parachlamydia from the water of an humidifier associated to an outbreak of fever

Birtles et al. Lancet 1997 Serological hint - in patients with community-acquired pneumonia

Marrie et al. Emerg Infect Dis 2001 - in patients with aspiration pneumonia

Greub et al. Ann NY Acad Sci 2003;990:311-319.

Greub G.

59 Greub –Intracellular bacteria in pneumonia

Parachlamydia and pneumonia

Epidemiological hint - Isolation of an amoeba containing Parachlamydia from the water of an humidifier associated to an outbreak of fever

Birtles et al. Lancet 1997 Serological hint - in patients with community-acquired pneumonia

Marrie et al. Emerg Infect Dis 2001 - in patients with aspiration pneumonia

Greub et al. Ann NY Acad Sci, 2003;990:311-319. Molecular hint - amplification of Parachlamydia DNA from lower respiratory tract samples (community-acquired pneumonia and bronchiolitis) Corsaro et al. Microbiology 2001, Greub et al. Emerg Infect Dis 2003. Ossewaarde et al. Microbiol 1999; Casson et al., J Clin Microbiol 2008. Greub G.

To further assess the role of Parachlamydia as an agent of pneumonia

Development of new diagnostic tools

Clinical samples from patients andwith withoutand withoutpneumonia, pneumonia,miscarriage, …

Parachlamydia N=224 PCR direct immunofluorescence

serology: seroconversion/IgM

2% of all patients

- 0% of patients without pneumonia - 4% of patients with pneumonia

Casson et al. In preparation

60 Greub –Intracellular bacteria in pneumonia

To further assess the role of Parachlamydia as an agent of pneumonia

Development of new diagnostic tools

Macrophages Parachlamydia Patients Pneumocytes

Animal model

Replication of Parachlamydia within mamallian cells

Human/murine macrophages Parachlamydia

Greub et al., Infection & 2003 Greub et al., Cell microbiol 2005 Lamp1 Croxatto et al, submitted Roger et al, in preparation Human pneumocytes (A549) Human lung fibroblasts (HEL)

Casson et al. Microbes and Infection 2006

To further assess the role of Parachlamydia as an agent of pneumonia

Development of new diagnostic tools

Macrophages Parachlamydia Patients Pneumocytes

Animal model

61 Greub –Intracellular bacteria in pneumonia

To further assess the role of Parachlamydia as an agent of pneumonia

Koch postulates

1. Bacteria may be detected in infected host 2. Bacteria may be isolated in pure culture from infected host 3. The same disease may be reproduced in animal 4. The same bacteria may be isolated in pure culture from experimentally-infected animals

Greub G.

Animal model •Intratracheal injection of 2.5 x 108 Parachlamydia acanthamoebae

Casson et al. Microbes & Infection, 2008

Animal model

100 Mice inoculated Mortality 90 with living Pac 80 Mice inoculated with 50% 5 days p.i. 70 heat-inactivated Pac 60 50 40

Survival (%) 30 20 10 0

0 1 2 3 4 5 6 7 8 9 10 Time (days post-infection)

Mice inoculated Weight loss 110 with living Pac (dead mice) Mice inoculated 100 about 30% with living Pac (surviving mice) 90 Mice inoculated with heat-inactivated Pac 80

70

60 Weight variation (%)

50

0 1 2 3 4 5 6 7 8 9 10 Time (days post-infection) Casson et al. Microbes & Infection, 2008

62 Greub –Intracellular bacteria in pneumonia

Histology: severe pneumonia

2 days post-infection Uninfected control

Purulent pneumonia in 100% of infected mice (days 2-4) Interstitial pneumonia (days 7-10)

Casson et al. Microbes & Infection, 2008

Presence of Parachlamydia in the lesions

40x 100x

LC

Casson et al. Microbes & Infection, 2008

Intra-nasal model J2 J10 Neg. control

Casson et al. In preparation

Anti-Parachlamydia Parachlamydia/macrophages

63 Greub –Intracellular bacteria in pneumonia

Conclusions

Parachlamydia acanthamoebae should be considered as a new agent of pneumonia

Further works are needed to better understand the biology of this obligate intracellular bacteria and to precise its pathogenic potential

Conclusions

Other Chlamydia-related bacteria might also be the agents of pneumonia of unknown etiology since they will remain undetected using axenic media

Better not to wait for the next outbreak…

Role of emerging intracellular bacteria in pneumonia

Role often suspected following an outbreak (intracellular bacteria may remain undetected)

Several agents yet to discover

Difficult to confirm a pathogenic role (commensals ?)

Aamoebae-resisting bacteria are good candidates i.e. Chlamydia-related bacteria

Useful to precise their mode of transmission and their antibiotic susceptibility

64 Greub –Intracellular bacteria in pneumonia

Research program

Clinical and environmental samples Estrella lausannenesis Amoebal co-culture Amoebal enrichment Cell culture Cell Criblamydia sequanensis permissiveness Antibiotic New species susceptibility

Taxonomy Biology Pathogenic role Animal model

Genomic and Development of new diagnostic tools Cell biology proteomic

Immunogenic proteins

Clinical samples from patients Adapted from: with and without pneumonia,pneumonia, miscarriage, … … Greub et al. Clin Microbiol Infect, 2009 Western -blot

Remerciements Antony Croxatto Claire Bertelli François Collyn Nicolas Gonzales Carole Kebbi Geneviève Goy Sébastien Aebi Julia Lienard

International collaborations: D Raoult, Marseille L Regan, London …

65