Educational Workshop EW10: Clinical Cases of Infections Caused by Intracellular Bacteria

Educational Workshop EW10: Clinical cases of infections caused by intracellular bacteria

Arranged with the ESCMID Study Group for Coxiella, Anaplasma, Rickettsia and Bartonella (ESCAR)

Convenors: A. Letaief (Sousse, TN) S.J. Cutler (London, GB)

Faculty: D. Raoult (Marseille, FR) A. Znazen (Sfax, TN) G. Greub (Lausanne, CH) – no handout available M. Wegdam-Blans (Veldhoven, NL) – no handout available

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Clinical cases of infections caused by intracellular bacteria 27/04/13 BERLIN Rickettsial infection • May 2012, in France

• Young girl (7 years)

• Tick bike after walking in wood

• The tick attached in the scalp

• A black lesion at site of tick bite

• A lymphadenopathy paiful in the neck

Cervical lymphadenopathy (left panel, arrow), inoculation on the scalp (middle panel), and residual alopecia 4 weeks later (right panel).

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• May 2012, in France

• Young girl ( 7 years)

• Tick bike after walking in wood

• The tick attached in the scalpscalp

• A black lesion at site of tick bite

• A lymphadenopathy paiful in the neckneck

WHAT IS IT?

• TIBOLA

Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks. Raoult D, Lakos A, Fenollar F, Beytout J, Brouqui P, Fournier PE. Clin Infect Dis. 2002 May 15;34(10):1331‐6

• DEBONEL [DEBONEL (Dermacentor‐borne‐necrosis‐erythemalymphadenopathy). A new tick‐borne disease?]. Oteo JA, Ibarra V. Enferm Infecc Microbiol Clin. 2002 Feb;20(2):51‐2. Spanish.

• SENLAT Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after Tick Bite. Angelakis E, Pulcini C, Waton J, Imbert P, Socolovschi C, Edouard S, Dellamonica P, Raoult D. Clin Infect Dis. 2010 Feb 15;50(4):549‐51. 5

. The tick: biting in winter/spring in the scalp

. The microorganisms

. How to manage

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DERMACENTOR TICKS IN EUROPE

Ambush strategy: • waits, falls • bites head • children • cold season • Females

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DERMACENTOR SEEKING HAIR

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Dermacentor reticulatus, the ornate dog tick

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D. marginatus, the ornate sheep tick

. The tick

. The microorganisms

. The disease

. How to manage

CAUSATIVE AGENTS 1‐ R.slovaca

A new tick‐transmitted disease due to Rickettsia slovaca. Raoult D, Berbis P, Roux V, Xu W, Maurin M. Lancet. 1997 Jul 12;350(9071):112‐3.

2‐ R.raoultii

Rickettsia slovaca and R. raoultii in tick‐borne Rickettsioses. Parola P, Rovery C, Rolain JM, Brouqui P, Davoust B, Raoult D. Emerg Infect Dis. 2009 Jul;15(7):1105‐8.

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R. slovaca infection (compared to MSF)

 Based on 17 PCR confirmed cases  More tick bite found  Lesion in the hair  Less fever  More cervical lymph node  Sequels : residual asthenia, localised alopecia  Low death rate  Young age  Females > males  Low serological titres

(Raoult D, et al. Clin Infect Dis. 2002 ;34:1331‐6.)

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Rickettsia slovaca infeco n

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 Cases very closely related to TIBOLA

 Clinical cases identical to that caused by R. Slovaca

 Present in Dermacentor, ¼ of that of R. slovaca

 Less pathogenic

 Present in all Eurasia 15

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Rickettsia slovaca and R. raoultii in tick‐borne Rickettsioses. Parola P, Rovery C, Rolain JM, Brouqui P, Davoust B, Raoult D. Emerg Infect Dis. 2009 Jul;15(7):1105‐8.

Abstract Tick‐borne lymphadenopathy (TIBOLA), also called Dermacentor‐ borne necrosis erythema and lymphadenopathy (DEBONEL), is defined as the association of a tick bite, an inoculation eschar on the scalp, and cervical adenopathies. We identified the etiologic agent for 65% of 86 patients with TIBOLA/DEBONEL as either Rickettsia slovaca (49/86, 57%) or R. raoultii (7/86, 8%).

Characteristics of TIBOLA/DEBONEL patients with certain or probable Rickettsia slovaca infection compared with patients with certain or probable R. raoultii infection*

The geographic distribution of these rickettsiae likely parallels that of Dermacentor ticks

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Rickettsia « la rioja »

Genetic characterisation of ompA, ompB and gltA genes from CandidatusRickettsia rioja. Portillo A, Ibarra V, Santibáñez S, Pérez‐Martínez L, Blanco JR, Oteo JA. Clin Microbiol Infect. 2009 Dec;15 Suppl 2:307‐8.

NOM : G Hôpital/Service : Hopital Fréjus BCU Prénom : G Sexe : F Médecin : Dr Pascal Del Giudice Date de naissance : Tel/Email : 04. 94 40 22 00 SYMPTOMATOLOGIE EPIDEMIOLOGIE Fièvre non  Voyage NON XEscarre unique / localisation : cuir chevelu  Escarres multiples / X Piqûre de tique / Identification morphologique: localisation : Dermacentor marginatus (femelle gorgée) Spectrométrie de masse: Dermacentor marginatus score 1.9 XAdénopathies / localisation : cervicales + douleurs ++ XPiqûre unique / localisation : cuir chevelu TRAITEMENT : Antibiotique / molécule: DOXY 4 jj PHOTO: XOUI  NON  2ème sérum demandé Culture: X Mis en culture le 08/11/2012 0 4 x10 A2 0:A2 MS Raw 4

3 Intens. [a.u.] 2

0 4 Coxie R. R. BioMol Date é chan s m ar lab lla_li Barto Rick sl ova c raoult ke a ii 07/11/2012 tique 1281736 pos neg 21/22 neg neg ecouv 1281734 neg neg neg croute 1281735 neg neg neg 22/11/2012 serum 1282695 neg Sérologie croute 1282691 neg

Date Smarlab Barto C. B Rick F. T Borrelia Diplo A.phago 08/11/2012 1212062 neg neg neg neg neg neg neg 22/11/2012 1212618 neg neg neg neg neg neg neg gltA gène: Rickettsia sp. DmS1 (591/592) AY129300 20 ompA gène: Candidatus Rickettsia rioja (452/453) EF028201, Rickettsia sp. Jl-02 (544/551) AY 093696

Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after Tick Bite. Angelakis E, Pulcini C, Waton J, Imbert P, Socolovschi C, Edouard S, Dellamonica P, Raoult D. Clin Infect Dis. 2010 Feb 15;50(4):549‐51.

Rickettsia slovaca and Rickettsia raoultii have been associated with a syndrome characterized by scalp eschar and neck lymphadenopathy following tick bites. However, in many cases, the causative agent remains undetermined. We report 3 cases of this syndrome caused by Bartonella henselae, and we propose the term "SENLAT" to collectively describe this clinical entity.

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1. NOM : D Hôpital de Nice, Dr. Pulcini BARTONELLA HENSELAE Prénom : B Sexe : F

DIAGNOSTIC

Adenopathie cervicale bilatérale Escarre cutanée : ulceration inflammatoire du scalp Tableau évoquant plutot un TIBOLA

Evolution à une semaine : l'évolution: persistance des ADP parotidiennes bilatérales (centimétrique à droite, 2 cm à gauche), fistulisation à la peau de l'ADP parotidienne gauche, avec tarissement spontané de l'écoulement puriforme en une dizaine de jours. Persistance d'une asthénie.

FACTEURS DE RISQUE

Contact avec animaux car vit en zone semi-urbaine Travaille dans 1 centre équestre : pense s’etre fait piqué par une tique ? Possède des chats

TRAITEMENT Bartonella Date Smarlab prélèvement Rickettsia sp. Bartonella sp. henselae 15 J de DOXY 09/04/2009 2961732 BCU neg 30,84/33,89 30,45/33,62

2. NOM : H Hôpital NANCY, Dr. Waton Prénom :Y Sexe : M

DIAGNOSTIC Adenopathie cervicale bilatérale Escarre cutanée du cuir chevelu PCR positive sur BCU à Bartonella henselae.

Francisella tularensis

Eschar and neck lymphadenopathy caused by Francisella tularensis after a tick bite: a case report. Edouard S, Gonin K, Turc Y, Angelakis E, Socolovschi C, Raoult D. J Med Case Rep. 2011 Mar 19;5:108.

INTRODUCTION: In 25 to 35% of cases, the aetiological agent of scalp eschar and neck lymphadenopathy after a tick bite remains undetermined. To date, Rickettsia slovaca, Rickettsia raoultii and more recently Bartonella henselae have been associated with this syndrome.

CASE PRESENTATION: Afour‐year‐old Caucasian boy was admitted to hospital with fever, vomiting and abdominal pain. On physical examination, an inflammatory and suppurating eschar was seen on the scalp, with multiple enlarged cervical lymph nodes on both sides. Although no tick was found in this scalp lesion, a diagnosis of tick‐borne lymphadenopathy was suggested, and explored by serology testing and polymerase chain reaction of a biopsy from the eschar. Francisella tularensis DNA was found in the skin biopsy and the serology showed titres consistent with tularaemia.

CONCLUSION: This is, to the best of our knowledge, the first reported case of scalp eschar and neck lymphadenopathy after tick bite infection caused by F. tularensis.

SENLAT tularémie infection à bartonella henselae

infection à Rickettsia slovaca 24

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. The tick

. The microorganisms

. How to manage ‐ Ticks  ‐ Serology ‐ Skin samples ‐ Treatment 25

Matrix‐assisted laser desorption ionization‐time of flight mass spectrometry for rapid identification of tick vectors. Yssouf A, Flaudrops C, Drali R, Kernif T, Socolovschi C, Berenger JM, Raoult D, Parola P. J Clin Microbiol. 2013 Feb;51(2):522‐8

A method for rapid species identification of ticks may help clinicians predict the disease outcomes of patients with tick bites and may inform the decision as to whether to administer postexposure prophylactic antibiotic treatment. We aimed to establish a matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS) spectrum database based on the analysis of the legs of six tick vectors: Amblyomma variegatum, Rhipicephalus sanguineus, Hyalomma marginatum rufipes, Ixodes ricinus, Dermacentor marginatus, and Dermacentor reticulatus. A blind test was performed on a trial set of ticks to identify specimens of each species. Subsequently, we used MALDI‐TOFMStoidentifyticksobtainedfromthewildor removed from patients. The latter tick samples were also identified by 12S ribosomal DNA (rDNA) sequencing and were tested for bacterial infections. Ticks obtained from the wild or removed from patients (R. sanguineus, I. ricinus, and D. marginatus) were accurately identified using MALDI‐TOF MS, with the exception of those ticks for which no spectra were available in the database. Furthermore, one damaged specimen was correctly identified as I. ricinus, a vector of Lyme disease, using MALDI‐TOF MS only. Six of the 14 ticks removed from patients were found to be infected by pathogens that included Rickettsia, Anaplasma, and Borrelia spp. MALDI‐TOF MS appears to be an effective tool for the rapid identification of tick vectors that requires no previous expertise in tick identification. The benefits for clinicians include the more targeted surveillance of patients for symptoms of potentially transmitted diseases and the ability to make more informed decisions as to whether to administer postexposure prophylactic treatment.

RAPID ID OF A TICK FROM PATIENT BY MALDI-TOF October 2-2012

Symtoms EPIDEMIOLOGY  Travel place / date :  Date of symtoms :  Fever X Morphological identification: Ixodes ricinus  Eschar only/ localisation :  Eschar multiples / localisation : X mass spectrometry: I.ricinus score 1,5 Adenopathy / localisation :  Single bite/ localisation :  multiple bite / localisation : Rash :  Maculo-papulus  Purpuric  Vesiculus  Animals contact: => Identify risk factor X Other manifestation: erythematous lesions

Date smarlab Bartonella C. burnetii Rickettsies Coxiella-like Borrelia A. phagoc 08/10/2012 1279089 neg neg neg neg neg ec 27

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NOM : L Hôpital/Service: Hôpital Nord/MIT Prénom : P Sexe : F Médecin : Dr Aubry Date de naissance: Tel/Email : 68933

SYMPTOMATOLOGIE EPIDEMIOLOGIE  Voyage lieu / date :  Date de début des symptômes : 01/12/2012 X Piqûre de tique / Identification : Dermacentor marginatus Fièvre NON Masse spectre : 1.837 Dermacentor marginatus X Escarre unique / localisation : cuire chevelu X Piqûre unique / localisation : cuire chevelu X Adénopathies / localisation : cervicale + lymphangite TRAITEMENT : Antibiotique / molécule: Doxy 200mg/j débuté le 01/12/2012 tout de suite après détection de tique

0 4 x10 G2 0:G2 MS Raw 3

Intens. [a.u.] 2

0 4

ARN R. R. Date prélèvement smarlab Barto C. B Rick F. T Borrelia 16S Ehr spiro C.like Diplo Actine 16S slovaca raoultii 05/12/2012 tique 1283793 neg neg 18/21 17/20 neg neg 35 sa ng 1283791 neg neg 21 écouvillon 1283792 neg neg neg 32/36 32/36 neg neg neg neg neg neg neg ec Date Smarlab Barto C. B Rick F. T Diplo 05/12/2012 1213183 neg neg neg neg neg

WB: négative

Culture tique Mis en culture le 05/12/2012- positive le 18/12/2012: Rickettsia slovaca

SKIN BIOPSY IS THE KEY OF DISCOVERY OF NEW RICKETTSIAL DISEASES

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SWABING Bechah Y, Socolovschi C, Raoult D. Identification of rickettsial infections by using cutaneous swab specimens and PCR. Emerg Infect Dis. 2011 Jan;17(1):83‐6.

Mouffok N, Parola P, Raoult D. Murine typhus, Algeria. Emerg Infect Dis. 2008 Apr;14(4):676‐8.

DIAGNOSTIC: PCR

Tested R. prowazekii B. quintana B. recurrentis

Ants 40 0 0 0

Lice 44 16 (36%) 5 (11%) 0

Tested gene Citrate synthase Citrate synthase 16srRNA ITS

Negative controls are critical Two different genes for confirmation of atypical/unique case Avoid “open” nested PCR and positive controls

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Antibiotics used in the literature in the case of SENLAT series (Scalp Eschar and Neck Lymphadenopathy after tick‐bite).

Auteurs N Doxycycline Macrolide (%) Autre traitement Aucun (%) a (%) traitement (%) Lakos 2002 (4) 8 16 (18,6) NP 45 (52,3) 14 (16,3) 6 Raoult et al. 2002 1 10 (71,5) Azythromycine : 01 (7,1) (14) 4 3 (21,4) Oteo et al. 2004 (6) 2 21 (95,5) Josamycine : 1 00 2 (4,5) Gouriet et al. 2006 1 13 (92,9) Josamycine : 1 00 (10) 4 (7) Ibarra et al. 2006 (7) 5 43 (79,6) Josamycine : 1 00 4 (1,9) Azythromycine : 10 (18,5)

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Performance for the Rickettsia spp. PCR. made from a skin swab the eschar or a skin biopsy of the lesion to the diagnosis of rickettsiosis.

Auteurs Na PCR Rickettsia positive PCR Rickettsia sur écouvillon cutané de positive sur l’escarre (%) biopsie cutanée (%) Wang JM et al. 2009 4 6/7 (85,7) NP (36) Bechah Y et al. 2011 9 8/9 (88,9) 5/5 (100) (33) Mouffok N et al. 2011 39 26/41 (63,4) 4/4 (100) (35) Renvoisé A et al. 2012 45 8/42 (19) 31/150 (20,6) (37)

14 Znazen - Bartonella infection

Abir Znazen, Adnene Hammami.

Laboratory of Microbiology, Habib Bourguiba University Hospital Sfax, Tunisia.

 1875: outbreak of Oroya Fever

 1885: Daniel Carrion (Peruvian medical student) inoculated himself with infected material

 Alberto Barton: discovery of the agent of Carrion’s Disease (Bartonella bacilliformis)

Historical overview Chronic Phase: Peruvian wart Vector: phlebotomine sand (Verruga Peruana) flies

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Historical overview

Bartonella henselae: Bartonella quintana: Cat scratch disease Trench fever

New species…………

 Taxonomy and genus description

 Epidemiology and transmission

 Clinical manifestations

 Case presentation: ◦ Bacillary angiomatosis ◦ Infective endocarditis

 Gram negative rods  Facultative intracellular  Slow growth on blood agar medium  Inert in most biochemical tests Limitation of Standard biochemical methods for identification  Molecular methods: Position of Bartonella species

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16S rRNA gene







17 Znazen - Bartonella infection

Rapidly growing number of Bartonella species and

genotypes

Multilocus typing and other molecular techniques

Full genome analyses of Bartonella strains appear to

be a more promising approach for developing a natural

typing system.

Natural cycle => Hemotropic life form: persistent intra-erythrocyte infection Specific mammalian host Transmission via arthropod vector

Harms A, CMR, 2012.

 Ubiquity

 Various animals : reservoir of zoonotic infections (pets++) ◦ Cats: B. henselae, B. clarridgeiae and B. koehlerae

 Well adapted host: asymptomatic bactereamia

 Seroprevalence: 14 to 50%

 Cat fleas: many species++, role in transmission ◦ Dogs: B.vinsonii. Subsp berkhoffi(endomyocarditis), B. henselae, B. clarridgeiae (endocarditis), B. washoensis, B. elizabethae, B. quintana and B. bovis ◦ Cattles: B. bovis (Europe, North America and Asia) ◦ Rats: B. elizabethae

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Table1: Bartonella species and their vectors and hosts Arthropod hosts Mammalian hosts Reservoir Incidental B. vinsonii subsp. berkhofii Tick dog Human B. vinsonii subsp. vinsonii Vole ear mite Vole B. vinsonii subsp. arupensis Tick Mouse Human B. taylorii ?Mouse, vole B. quintana Body louse Human B. henselae Cat flea, tick Cat Human B. koehlerae ?Cat Human B. alsatica ? Rabbit Human B. grahamii Fleas Mouse, vole Human B. elizabethae ?Rat Human B. tribocorum ?Rat B. birtlesii ?Mouse B. doshiae ?Vole B. rochalimae ?Dog, fax B. clarridgeiae ?Cat B. bovis ? Cattle B.capreoli ? Roe deer B.chomelii ? cattle B.schoenbuchensis Deer ked Roe deer B. bacilliformis Sand flies Human

B. bacilliformis B. quintana B. henselae

Homelessness alcoholism Poverty Poor conditions of hygiene Body louse

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Other Bartonella species

B. vinsonii subsp. berkhoffii B. grahamii B. doshiae B. vinsonii subsp. Berkhoffii B. alsatica B. elizabethae B. clarridgeiae B.myotimonensis B. koehlerae B. washoensis B. tamiae B. koehlerae

B. bacilliformis B. quintana B. henselae

•Carrion •Trench fever •Cat Scratch Disease •Bacillary disease angiomatosis •Bacillary •Infective angiomatosis endocarditis •Infective endocarditis

Table3: clinical presentation associated with Bartonella sp

species Human disease B. elizabethae Endocarditis, neuroretinitis B. clarridgeiae CSD, sepsis, endocarditis B. koehlerae Endocarditis, CSD B. vinsonii subsp. berkhoffii Endocarditis, arthralgia / myalgia / headache /fatigue B. washoensis Fever and myocarditis B. tamiae Fever B. grahamii Neuroretinitis B. doshiae CSD B. alsatica Endocarditis, lymphadenitis

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B. henselae pathogenesis

Cat scratch disease

Intra-erythrocytic Infective bactereamia endocarditis

Peliosis Hepatitis Chronic bactereamia

Bacillary angiomatosis

Ctenocphalides felis

B. quintana pathogenesis

 Trench fever  Chronic bactereamia  Bacillary angiomatosis  Infective endocarditis+++

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 50 year old women  Immunocompetent  Djbeniana (rural area)  Low socioeconomic level  Multiple angiomatosis lesions persisting more than 6 months.

Figure: angiomatosis lesions localized on the left hand

Tunisia Sfax

Diagnosis

Histological lesions:+++

Hematoxylin eosin staining Warthin starry staining

Microbiology: ITS-PCR: Bartonella quintana

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Bacillary angiomatosis

Treatment

 Erythromycin: 3g/day during 3 months  Outcome: favorable

 61 year old men  Immunocompetent  Sfax (Awabed locality)  Continuous fever  Low socioeconomic  Dyspnea status (hygiene)  Abdominal pain  No history of any cardiac disease

Patient’s characteristics Clinical features

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 Fever: 39°

 BP: 100/40mmHg, HR: 84bpm, RR: 20C/mn

 Systolic and diastolic murmur in the mitral and aortic areas

 Chest X ray: infiltrates

 Abdominal echography: hepatomegaly

 CRP: 144mg/l

 ESR: 70 mm

 WCC: 17120/mm3, hb: 8.2g/dl, Pl: 65000/mm3

 Liver enzymes: 33/19 IU/l

 3 sets of blood cultures : negative

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ETT: performed march 14 2013: vegetations on mitral and aortic valves + calcifications

 ETO (March 15 2013):

◦ Aortic valve: mobile vegetation (20mm)

◦ Mitral valve: many vegetations with

calcification+ perforation

◦ Perivalvular abscess

Diagnosis: infective endocarditis

Geographic origin

Poorness

Defective hygiene

Destructive valve lesions

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Znazen A et al, AJTMH, 2005

 The clinical and pathological characteristics

of Bartonella endocarditis :

◦ Previous history a valvular disease :missing

◦ More often affecting the aortic valve

◦ Highly vegetative lesions with calcification

Raoult D, JAMA int Med,2003

Serology: Coxiella burnetii Bartonella Brucella Secondly: Chlamydia, Legionella, Mycoplasma…. PCR on whole blood: Standard PCR/ RT-PCR+++ Genes: ITS, rnpb, fur, pap, Groel…..

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Serology results Chlamydia Bartonella Coxiella burnetii pneumoniae quintana/henselae Phase I /II IgT - 6400/3200 - IgG 2048 -Neg IgM Neg - Neg IgA Neg - Neg • What is your diagnosis?

Bartonella endocarditis Cross reactions between Chlamydia and Bartonella +++

 Previously : almost all cases of Chlamydia endocarditis were in fact Bartonella endocarditis. (Maurin M , JCM, 1997)  In our series of BCNE: 13 patients with Bartonella IE showed positive serology to three chlamydial species (Znazen A , AJTMH, 2005)

 Micro immunofluorescence assay:

◦ Gold standard

◦ Cross reactions between species

 Western blott with cross adsorption:

◦ To confirm species

◦ Antigen consuming

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Fournier PE, CDLI, 2002 IgG titer> 1:800: .PPV of 0.81in patients with chronic infection .PPV of 0.955 in patients with infective endocarditis

Clinical case (continues):

Patient was operated on March 22 2013

Prosthetic Aortic and mitral valves

. Culture . PCR . Histology . Immunohistochemistry

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Antimicrobial drug susceptibility of Bartonella :

 Penicillin  Cephalosporin  Aminoglycosides Raoult D, JAMA int Med,2003  Chloramphenicol  Doxycycline, tetracycline  Macrolides  Rifampin  Fluoroquinolones  cotrimoxazole

Rolain JM, AAC, 2004

 Aminoglycosides: 14 days

 Recommended antibiotherapy:

◦ Doxycycline: 100mg*2/day for 6 weeks

◦ Gentamicin : 3mg/kg/day for 2 weeks

 Bartonella species: several host reservoirs ◦ B.quintana: detected in fleas!

 Wide clinical spectrum

 Diagnosis: Serology / PCR++

 MLST + complete genome sequences: ◦ Natural history : reservoir‐transmission‐IE

 Bartonella Network : ◦ Better description of epidemiology and pathogenicity

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 Cardiology department, Hedi Chaker university hospital

 Infectious diseases department, Hedi Chaker university hospital

 Laboratory of Micrbiology , Habib Bourguiba university hospital