GIKAS ESCAR-2010final.ppt [Kompatibilitätsmodus]

Educational WorkshopEW15: Infections by intracellular pathogens: from the laboratory to the clinic

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

Convenors: Gilbert Greub (Lausanne, CH)( , )Achilleas Gikas (Heraklion, GR)

Faculty: Achilleas Gikas (Heraklion GR)Faculty: Achilleas Gikas (Heraklion, GR)Pierre-Edouard Fournier (Marseille, FR)Gilbert Greub (Lausanne, CH)Jean-Marc Rolain (Marseille, FR)

Gikas - Evolution of serology

A.Gikas Prof. Int.Medicine-Infectious Diseases

Infections by intracellular pathogens: from the l b h li i

ΠΑΝΕΠΙΣΤΗΜΙΟ ΚΡΗΤΗΣ

UNIVERSITY OF CRETE

ΤΜΗΜΑ ΙΑΤΡΙΚΗΣ

FACULTY OF MEDICINE

laboratory to the clinic

Topic 1 Evolution of serology

History of Patient with Fever -Headache - Rash

♂♂ 41 y.o – French – Medical history (-)For holidays in a trailer camp in South Crete since

8/7/03

12/7/03 neck pain headache ulcerated painless12/7/03 neck pain, headache, ulcerated painless nodule on dorsal surface of Right 1st MTT-phalangeal joint

16/7/03 fever 39.2° C with shivers, lower limb myalgia, frontal headache enlarged (~1.5cm) & slightly painful inguinal lymph node (Right)Physician CF2G

3


Presentation in hospital 20/7/03 - Day820/7/03 Dorsal surface of Right 1st MTT-phalangeal

joint : Reddish, hard, painless nodule ~15mm with central ulceration (~3mm) and brown crust

Maculopapular hyperaemic rash on torso&limbs,including soles, palms, face.

Remaining physical exam normalRemaining physical exam. normal

Main lab abnormalitiesSGOT/SGPT 77/87 LDH 594ESR 25

Differential diagnosis?Differential diagnosis?1. Viral: Coxsackievirus A9Echovirus 9 Epstein-Barr virus Cytomegalovirus

Parvovirus B19 Measles Rubella

Question 1

Parvovirus B19 Measles Rubella Chickenpox

2. Typhoid fever3. Erythema multiforme4. Idiopathic thrombocytopenic purpura (ITP)5. Mediterranean Spotted Fever (MSF)

Which laboratory test would you choose Which laboratory test would you choose if you suspected rickettsial infection if you suspected rickettsial infection ??

1.Weil–Felix test2.Complement fixation test

Question 2

3.Enzyme-linked immunosorbent assay (ELISA)

4.Indirect immunofluorescence test (IFAT)

5. Western immunoblot / Cross-Absorption assay

4


IFAT Antibodies 22/7/03 25/7/03 30/7/03Rickettsia conorii IgM 1/100 1/200 1/1600

IgG 1/60 1/240 1/1920

IgA (-) 1/400 1/1600

Rickettsia typhi IgM (-) (-) 1/100IgG (-) (-) (-)

IgA (-) (-) (-)

Coxiella burnetii IgM (-) (-) (-)IgG 1/60 (-) 1/60

IgA (-) 1/400 (-)


IgG 1/60 1/240 1/1920

IgA (-) 1/400 1/1600

Rickettsia typhi IgM 1/600 1/200 1/1600IgG 1/120 1/240 1/120

IgA (-) 1/400 1/800

Coxiella burnetii IgM (-) (-) (-)IgG 1/60 1/120 1/960

IgA (-) 1/400 (-)

1.Weil–Felix test

2.Complement fixation test

Which serologic method is considered the reference Which serologic method is considered the reference method for diagnosis of rickettsioses when method for diagnosis of rickettsioses when crosscross--reactions occur?reactions occur?

Question 3

3.Enzyme-linked immunosorbent assay

4.Indirect immunofluorescence test (IFAT)

5.Indirect immunoperoxidase assay

6. Western immunoblot assay / Cross-Absorption Assay

5


Which therapeutic regimen would you Which therapeutic regimen would you administer?administer?

1. Amoxicillin 2g/Day

Question 4

2. Ciprofloxacin 1500 mg/Day

3. Doxycycline 200 mg/Day

4. Chloramphenicol 2 g/Day

Treatment – Clinical course

22/7/03 tab Doxycycline 100 mg 1×2

24/7/03: Subjective improvement. Rash has started remittingConsiderable improvement of headache

25/7/03: Good general condition, Defervescence.Further improvement of rash.

26/7/03: Discharge under antibiotic therapy & instructions.

30/7/03 (Follow-up) Good general condition. Rash has completely subsided.

TAXONOMYKingdom - Procaryotae

Superkingdom - The BacteriaPhylum - Proteobacteria (alpha subdivision)

Order - Rickettsiales ("Rickettsiae")

Family -RickettsiaciaceaeGeneraGenera

RickettsiaOrientia

Family Anaplasmataceae,

GeneraEhrlichia, Anaplasma, Neorickettsia, and Wolbachia

6


Obligate intracellular parasites are parasitic microorganisms that cannot reproduce outside their host cell, forcing the host to assist in the parasite's reproduction.

Obligate intracellular parasites of humans include:VirusesCertain bacteria, including: Chlamydia, and closely related species

Obligate intracellular parasites Obligate intracellular Pathogens

Rickettsia Anaplasmata CoxiellaCertain species of Mycobacterium such as Mycobacterium lepraeCertain protozoa, including: Plasmodia speciesLeishmania spp.Toxoplasma gondiiTrypanosoma cruzi

Study of obligate pathogens is difficult because they cannot usually be reproduced outside the host.

sDiagnostic Strategy of Rickettsioses and Ehrlichioses

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

SEROLOGICAL METHODSThe easiest methods for the diagnosis of rickettsial diseases

• Weil–Felix test

• Complement fixation tests,

• Enzyme-linked immunosorbent assay

• Indirect immunoperoxidase assay

• IFA test• Microimmunofluorescence (MIF)• Cross-Adsorption Assay/ WB immunoassay

7


Weil–Felix test1915 by bacteriologists Edmund Weil and Arthur Felix

It is used to diagnoserickettsioses based on serological cross-reactions

Antibody reactions:• P. vulgaris 0X19 identifiesP. vulgaris 0X19 identifies Typhus group (TG) rickettsiae (R. prowazekii and R. typhi) and R. rickettsii, • P. vulgaris 0X2 identifiesSpotted fever group (SFG) rickettsiae • P. mirabilis OXK identifies Orientia tsutsugamushi

The antibodies detected are IgM produced for only a short time following infectionLacks sensitivity and specificity Kaplan JE, et al. Am J Trop Med Hyg 1986

Amano K. et al. JCM 1992La Scola B, Raoult D. JCM 1997

Very insensitive in the first3 weeks post-onset

Strongly IgG dependent

Labor intensive

Complement fixation test

Labor intensive

Requires substantial quantities of antigen

Lacks sensitivity and specificity

Actually not recommendedCimolai N (ed). Marcel Dekker Inc, NY, USA, 2001.

Shepard CC, et al. JCM 1976

Newhouse VF, et al. Am J Trop Med Hyg; 1979

First introduced for detection of antibodies against R. typhi and R. prowazekii

Highly sensitive and reproducible

ELISA

Highly sensitive and reproducible

Differentiation of IgG and IgM antibodies

Later adapted to the diagnosis of RMSF and other rickettsioses

Clements ML et al. J Infect Dis 1983Dobson ME et al. Am J Med Hyg 1989

8


The indirect immunoperoxidase assay

• Uses the same antigen but requires only light microscopy (uses peroxidase instead of fluoroscein) – an important economical factor for developing countries

• As easy to perform as the IFA test

• As rapid as the IFAT

• As sensitive and specific as the IFAT

Raoult D et al. Eur J Clin Microbiol; 1985

Kelly DJ, et al. Am J Trop Med Hyg; 1988

Walker DH, et al. Lab Invest 2000

The indirect immunofluorescence assay (IFA)

The reference serological test

• Determination of both IgG and IgM antibodies

• Early and late or convalescence phase serum: 15 days

• Seroconversion: fourfold or greater increase in antibody titers

Cut-off values (Rick)Different in other laboratoriesIFA is highly sensitive May lack specificity

R. rickettsii

Patient IgG

Anti-human IgG-FITC


Cross-reactions withRickettsiae and 1.Proteus, Legionella, Bartonella, and Ehrlichia infections 2. within TG and within the SFG

False-positive IgM with:

The indirect immunofluorescence assay (IFA)

p g1.The rheumatoid factor 2.Infections generating unspecific lymphocyte B proliferationviral and parasitic

Cytomegalovirus,Epstein–Barr virus, Malaria).

Cross-reactions could also be observed between HME and HGA.


9


Simultaneously detects antibodies to several antigens with the same drop of serum in a single well containing multiple dots

Tests for several antigens on the same slide may allow the causative agent to be identified from its comparatively higher antibody level

Microimmunofluorescence (MIF)

higher antibody level

Rolain JM et al. Clin Diagn Lab Immunol 2003

R. africae

R. australis

R. conorii

R. rickettsii

R. typhi

C. burnetii

Serological diagnosisAcute infection Chronic infection / Endocarditis

C. burnetii • anti-II IgM ≥1:50 or anti-II IgG ≥1:200• 4X antibody increase in paired sera

anti-I IgG ≥ 1:800

Bartonella spp 1:50 ≤ IgG ≤ 1:400 (Se varies depending on method)

• Chronic bacteremia IgG ≤1:800 • Endocarditis IgG ≥1:800 (97% PPV)

R. conorii • IgG ≥1:128 and/or IgM ---R. conorii gG 8 a d/o g≥1:64 are indicative• 4X antibody increase within 2-week interval

R. typhi and other rickettsioses

• IgG ≥1:64 and/or IgM ≥1:32 are suggestive • 4X antibody increase within 2-4 week interval

---

HME (E. chaffeensis)HGA (A. phagocytophilum)

• IgM ≥1:32 • 4X antibody increase in paired sera

--- Wilkinson HW et al. J Infect Dis 1983Dupont HT et al. Clin Diagn Lab Immunol 1994

Fournier PE, et al. Clin Diagn Lab Immunol; 2002Brouqui P et al. CMI 2004

Gouriet F, et al. Clin Microbiol Infect; 2008Raoult D, Parola P, eds. Informa healthcare 2008

Specific diagnosis in case of cross-reactions

1. SEROLOGY :IgG and/or IgM antibody against an antigen > two serial dilutions of IgG and/or IgM antibody against other rickettsial antigens


IgG 1/60 1/240 1/1920

IgA (-) 1/400 1/1600

Rickettsia typhi IgM (-) (-) 1/100IgG (-) (-) (-)

IgA (-) (-) (-)

Coxiella burnetii IgM (-) (-) (-)IgG 1/60 (-) 1/60

IgA (-) 1/400 (-)

10


2.2. WB IMMUNOASSAY:WB IMMUNOASSAY:When titers between antibodies are lower than two dilutions

A rickettsial antigen is the agent of the infection ifacute or convalescent sera show an exclusive reactivity with the specific proteins antigens of this antigen only

Teysseire N, Raoult D. JCM 1992La Scola B, Raoult D. JCM 1997

Houpikian, Raoult. Clin Diagn Lab Immunol 2002

B. quintana

B. henselae

B. vinsonii

B. elizabethae

3.3. CROSS ABSORPTION STUDYCROSS ABSORPTION STUDY:

Used to discriminate cross-reactions between ≥2 antigens sharing common epitopes

Is performed when WB immunoassays are not diagnostic

Requirement: IgG/IgM must be ≥ 128/32Requirement: IgG/IgM must be ≥ 128/32

After cross-absorption studies confirming the agent(i) IF serology positive for a single antigen, or(ii) WB immunoassay showing an exclusive reactivity with

specific proteins of a sole agent

Very expensive and time-consuming


Cross-Absorption Assay

1. The serum is mixed separately with eachbacterium involved in the cross-reaction, e.g. R. conorii and R. ascelmanii

2. Then the serum is tested against each of these antigens to absorb the corresponding antibodies

3. Disappearance of only heterologous antibodies bacterium responsible for cross-reaction

4. Disappearance of both homologous & heterologous antibodies bacterium responsible for disease

La Scola B, et al. Clin Diagn Lab Immunol; 2000

11


Due to Due to common Ag epitope (red)common Ag epitope (red)ppatient’s serum shows crossatient’s serum shows cross--reaction reaction with Ag1 & Ag2with Ag1 & Ag2

IFAIFA

Absorption of patient’s serum with Ag01 Absorption of patient’s serum with Ag01

Ag1Ag1

Ag2Ag2

Ag1Ag1

WBWB

Absorption with R. conorii

Absorption of patient’s serum with Absorption of patient’s serum with Ag02 eliminates Ag02 eliminates bothboth heterologous & heterologous & homologous antibodieshomologous antibodies

Bacterium responsible for diseaseBacterium responsible for disease

p p gp p geliminates eliminates onlyonly heterologous antibodiesheterologous antibodies

Bacterium responsible for crossBacterium responsible for cross--reactionreaction


Ag2Ag2Absorption with R. ascelmanii

Automation of serodiagnosis with the Multiplexed automated corpuscular antigenic microarray (MACAM)• Miniaturization of IFAT (1 nL antigen sol./slide vs 1 μL for IFAT)

• One serum specimen can be tested simultaneously for multiple pathogens

• Applied to all the bacteria known to cause a defined clinical syndrome, e.g. culture-negative endocarditis

• Totally automated platform similar to those of the ELISA

• Four internal spots to control six steps of the serology (interpretation independent of investigator)

• Digitization of fluorescent spots

• Much less expensive

• The highest level of reproducibilityGouriet F, et al. Clin Microbiol Infect; 2008

http://www.inodiag.com/inodiag_incubateur.asp# accessed March 11th 2010

Automation of serodiagnosis with the Multiplexed automated corpuscular

antigenic microarray (MACAM)

Gouriet F, et al. Clin Microbiol Infect; 2008

12


Automation of serodiagnosis with the Multiplexed automated corpuscular antigenic microarray (MACAM)

Coxiella burnetii, Chlamydophila pneumoniae, Chlamydia psittaci, Legionella pneumophila, Francisella tularensis, Mycoplasma pneumoniae


Multiplexed automated corpuscular antigenic microarray (MACAM)

Algorithm of interpretation for blood negative-culture endocarditis


Other Diagnostic approaches (1)Phage display library

• Identifies bacterial epitopes

• Epitope libraries displaying random peptides are used to locate functional determinants or epitope sites of microbial antigens

• random peptides• random peptides are displayed on the surface of the phage particles

Naidu BR et al. Immunol Lett; 1998

13


Other Diagnostic approaches (2)

High Throughput Sequencingand Proteomics to rapidly Identify Immunogenic Proteins of a New Pathogen

Or any pathogen for which better diagnostics are needed

To elaborate the first step of an ELISA test

Greub G et al. PLoS ONE 2009

2D map and identification of P. acanthamoebae immunogenic proteins

Future meetings of ESCAR:Future meetings of ESCAR:

ESCAR PostESCAR Post--graduate educational course “Intracellular Bacteria: graduate educational course “Intracellular Bacteria: from Biology to Clinic”, from Biology to Clinic”, Sousse, TunisiaSousse, Tunisia, 2, 2--5 November 2010 .5 November 2010 .

66thth International Meeting on Rickettsiae and Rickettsial International Meeting on Rickettsiae and Rickettsial diseases, diseases, Heraklion, Greece,Heraklion, Greece, 2011.2011.

[email protected]

14

Fournier - Genomics and clinical application

2020thth ECCMID, Vienna 2010ECCMID, Vienna 2010

GENOMICS AND CLINICAL GENOMICS AND CLINICAL APPLICATIONSAPPLICATIONS

PierrePierre--Edouard FournierEdouard Fournier

Unité des Rickettsies et Pathogènes émergents Unité des Rickettsies et Pathogènes émergents URMITE, CNRSURMITE, CNRS--IRD UMR6236, IRD UMR6236,

Faculté de Médecine, Université de la MéditerranéeFaculté de Médecine, Université de la MéditerranéeMarseille, FranceMarseille, France

UR

UR

1,010 bacterial genomes as of Feb. 261,010 bacterial genomes as of Feb. 26thth, 2010, 2010

169

185

213

150

200

250

2 2 4 5 413

25 29

4859

80

137

35

0

50

100

150

Candidatus Candidatus ‘’Carsonella ruddii’’‘’Carsonella ruddii’’0.15 Mb0.15 Mb

Sorangium cellulosumSorangium cellulosum13 Mb13 Mb

15


>200 genomes from human pathogens>200 genomes from human pathogens

40 species > 1 genome (2 to 12)40 species > 1 genome (2 to 12)40 species > 1 genome (2 to 12)40 species > 1 genome (2 to 12)

Empirical choiceEmpirical choice rational choicerational choice

Phenotype predictionPhenotype prediction

blaVEB-1 aadB arr-2 cmlA5 blaOXA-10 aadA1

1_1791_1761_1661_1651_163 1_173

blaVEB-1 aadB arr-2 cmlA5 blaOXA-10 aadA1Culture media

design

Predictionof virulence

Prediction of antibioticresistance and

antibiotic design

Molecular detectionMolecular detectionIdentificationIdentification

ATGTTCACAAGGACTATCATGAACATCGGCTATGTTGATGATGTACAACCTTTAAAACAGGGAGTACGTT TAAATTTTTCTACGCGCTATGACATACAGAGTTTGGAAATTGGTGCATCGATTGCGTGTTCATGGATTTG TCTGACAATTGTCGAGCGGGGGGTAAAACAAGCGACTGCTGGTTGGTTTGCTGTAGAAGCATGGGAAGAA GCATTGCGTTTGACTAATCTTGCACAATGGACAAAAGGAACTTTTGTTAATTTGGAACGTTCGCTTCGAT TAGGTGATGAAATAGGAGGACATTTGGTTTCCGGTCATATTGATGGTTTGGCTGAAATCATTGATCAAAA AAATGAAGGGGATGCAATTCGTTTTTATTTAAAAGTTGTAAGACAATTTATGCCTTTCATTGTCAATAAG GGATCTATTGCACTTAATGGGACATCTTTGACTGTTAATGGTGTTGAGGATTGTGTTTTTGATGTTCTTA

TAAAGTTTTTAAGCCATTTTTTAAAGCTTTACATAAAGTTTTTAAGCCCTCTTTTAAAGCATTGCATAAAGTTTTTAAGCCATCTTTTAGAGGTTTACATAAAGTTTTTAAGCCATCTTTTAAAGCTTCACA

DNA micro-arrays

Prediction of restriction profiles

Identification of specific signatures

Vaccine and serologicalVaccine and serologicaltest developmenttest development

TTATTCGCCATACACTCGAAATGACAACGTGGGGACAAGCTAGAATTGGAGATTGGGTCAATTTGGAAAT TGATCAACTTGCTCGTTATGCTGCGAAACTTTTTGCTTTAAAAAGAGAAGATGAATAA

Identificationof antigenic epitopes

Vaccine designDevelopment of monoclonal

antibodies

Molecular detection & identificationMolecular detection & identificationMolecular detection & identificationMolecular detection & identification

16


Two objectivesTwo objectives

Detection Detection -- IdentificationIdentification


GenotypingGenotyping

Genome comparisonGenome comparison

Rational target selectionRational target selection

•• GenusGenus-- or speciesor species--specific fragmentsspecific fragments•• Multiple copies for increased sensitivityMultiple copies for increased sensitivity•• Most conserved or variable sequencesMost conserved or variable sequences

Aim 1 = detect any bacterium Aim 1 = detect any bacterium => select “universal” genes => select “universal” genes (bacterial core genes)(bacterial core genes)

Koonin EV. Comparative genomics, minimal gene-sets and the last universal common ancestor. Nat. Rev. Microbiol. 2003;1:127-136.

17


Aim 2 = detect specific bacteriaAim 2 = detect specific bacteria=> select => select genus or speciesgenus or species--specific sequencesspecific sequences

Colson et al. SVARAP and aSVARAP: simple tools for quantitative analysis of nucleotide and amino acid variability and primer selection for clinical microbiology. BMC Microbiol. 2006;6:21.

•• ObjectiveObjective == increaseincrease PCRPCR sensitivitysensitivity withoutwithout losinglosingspecificityspecificity

•• NestedNested PCR,PCR, singlesingle--useuse targettarget andand primers,primers, nono positivepositivecontrol,control, numerousnumerous negativenegative controlscontrols

•• SelectionSelection ofof primersprimers byby aligningaligning closelyclosely--relatedrelated genomesgenomes

Aim 3 = increase detection sensitivityAim 3 = increase detection sensitivity=> “Suicide” PCR=> “Suicide” PCR

•• SelectionSelection ofof primersprimers byby aligningaligning closelyclosely relatedrelated genomes,genomes,inin conservedconserved zoneszones flankingflanking aa variablevariable fragmentfragment

•• SignificativelySignificatively moremore sensitivesensitive thanthan usualusual PCRPCR andandcultureculture

•• Specimens: blood, serum, skin biopsies, arthropods

Raoult Raoult et alet al. Proc. Natl. Acad. Sci. USA. 2000;97:12800. Proc. Natl. Acad. Sci. USA. 2000;97:12800--3.3.

18


Aim 3 = increase detection sensitivityAim 3 = increase detection sensitivity=> Target multi=> Target multi--copy sequencescopy sequences

•• SignificativelySignificatively moremore sensitivesensitive thanthan usualusual PCRPCR ((pp == <<1010--22))

• Applicable to all specimens

Aim 4 = identification / phylogenyAim 4 = identification / phylogeny=> select a target according to desired taxonomic level=> select a target according to desired taxonomic level

Fenollar and Raoult. APMIS,112:785Fenollar and Raoult. APMIS,112:785--807, 2004807, 2004

Identification / PhylogenyIdentification / Phylogeny

•• sca1 sca1 = 1,8 = 1,8 –– 5,9 kb5,9 kb•• Only Only sca sca gene present in all gene present in all Rickettsia Rickettsia speciesspecies•• AutoAuto--transporter => double selection pressuretransporter => double selection pressure•• 488488--bpbp--fragment identifies all species fragment identifies all species (F1MAX(F1MAX--RMAX)RMAX)

19



Detection Detection -- IdentificationIdentification


GenotypingGenotyping

Why typing bacteria ?Why typing bacteria ?

2 main objectives

• Tracing strains responsible for outbreaks within an hospital or a local community: growing need tohospital or a local community: growing need to identify strains of bacterial pathogens with increased virulence, transmissibility, antibiotic-resistance, or involved in bioterrorist attacks

• Global or long term epidemiology: monitoring how microbial populations change over time

•« Ultimate genotyping method »

Complete genome sequencingComplete genome sequencing

• As yet unadapted to strain typing

20


Non sequenceNon sequence based methodsbased methodsNon sequenceNon sequence--based methodsbased methods

A

B

1 1’ 2 2’

2’21’1

P i 1 1’ lifi ti

VNTRVNTRVariable number of tandem repeatsVariable number of tandem repeats

Genome sequences => rational selection of tandem repeatsGenome sequences => rational selection of tandem repeats

Primer 1 - 1’

2 - 2’

amplification

Electrophoresis1 2

A - B A - B

SNPSNPSingle nucleotide Single nucleotide

polymorphismpolymorphism

21


MicroarrayMicroarray

Microarray 3,875 genesMicroarray 3,875 genes

Highly discriminant Highly discriminant butbutwill identify only will identify only missingmissing

genesgenes

T. whippleiT. whipplei microarraymicroarray

SequenceSequence based methodsbased methodsSequenceSequence--based methodsbased methods

22


• Most variable sequences between genomes from closely related bacteria = most variable among strains of a species

• Non-coding (intergenic) sequences are more variable h di (l l i )

HypothesesHypotheses

than coding sequences (less selection pressure) (Dobrindt et al. (2001) Curr. Opin. Microbiol. 4, 550-7)

• Most studied spacer = 16S-23S rRNA, great variability in sequence, length and number of alleles but not present in all bacteria (Rickettsiales)

MultiMulti--spacer typing (MST)spacer typing (MST)

• In silico search for the most variable intergenicsequences between 2 closely-related genomes(strains or species)

• Amplification / sequencing• Amplification / sequencing

• 1 unique sequence = 1 genotype

• Combination of genotypes obtained fromintergenic sequences = multispacer typing (MST)

Rational choice of target sequencesRational choice of target sequences

R. prowazekii genome

R. conorii genome

5’

5’ 3’

3’

Conserved genesDegraded genes (split or fragment)Conserved spacers (BLASTN score > 75)

Variable spacers (BLASTN score < 75)

23


Creation of the free-access, online MST-Rick database(http://ifr48.timone.univ-mrs.fr/MST_BHenselae/mst).


Culture medium designCulture medium design


Antibiotic resistanceAntibiotic resistance Detection of virulenceDetection of virulence

Source of information Source of information for metabolismfor metabolism

• Several essential aminoacid pathways are lacking

24


Folate Folate biosynthesis biosynthesis and enzymesand enzymes

Dihydropteroate synthase y p y(DHPS)

Sulfamethoxazole target

Dihydrofolate reductase (DHFR)

Trimethoprim target





25


5’ partialATPase

1_837

strA

1_59

strB

1_62

dhfrI

1_73

sulI cmlA tetR tetA

1_81 1_88 1_748 1_103

aac6’

1_126

sulI

1_203

dhfrX

1_196

sulI

1_187

blaVEB-1 aadB arr-2 cmlA5 blaOXA-10 aadA1

1_1791_1761_1661_1651_163 1_173

pbrR Heavy metaldetoxification protein

1_808 1_43

qacEΔ1

1_78

qacEΔ1

1_199

qacEΔ1

1_183

Mercury resistance operon

arsH arsB arsC arsR arsC

Arsenic resistance operon

1_817 1_816 1_814 1_813 1_812

ISPpu12-like transposon truncated Tn5393-liketransposon

intIintegrase

1_779

GroEL-intégrasefusion protein

1_724

intIintegrase

1-703

recG

1_718

trkAputative

monooxygenase

trxBthioredoxinreductase

1_821 1_819

uspA

1_811

lspA

1_44

lysR

1_107

orf5

1_207

tniA Transpositionhelper

1_16 1_17

Transposase

1_52

Transposase-like protein

1_732

Transposase

1-710

TransposaseORF513

1_194

IS1999

1_699

IS6100includes tnpA

1_633

1_780

tnpM

3’-partialATPase

1_434

Other bacteria

Best Blast match with

Pseudomonas sp.Salmonella sp.

E. colisulI

1_442

aphA1

1_522

aac3

1_339

aadDA1

1_348

tetR tetA

1_598 1_258

cat

1_569

sulI

1_356

pbrR Heavy metaldetoxification protein

1_465 1_376

qacEΔ1

1_352

merE merC merP merT merRmerD merA

1_6121_6151_6211_6221_624 1_2441_609

ISPpu12-like transposon

Tn21-like transposon truncated Tn1721-like transposon IS1-like transposon

intIintégrase

1_513

Résolvase

1_36

5

1_366

trbI

1_467

lspA

1_377

Résolvase

1_318

pecM

1_588

ybjA

1_277

orf5

1_360

orfX

1_343

orfX’

1_344

orfX

1_341

Transposase

1_386

InsB (Tn1)

1_287

IS15

1_562

Transposase

1_555

Transposase

1_314

Transposase Transposase

1_528 1_319

TnpA

1_213

urf2y

1_627

TnpA

1_275

InsA (Tn1)

1_285

tnpM

1_516

Transposase

1_326





Identification of virulence markersIdentification of virulence markers

Whole genome sequencing of meticillin-resistantStaphylococcus aureusMakoto Kuroda, et al. Lancet 2001; 357: 1225–40

Pathogenicity islandsPathogenicity islands

26


Serological test and vaccine developmentSerological test and vaccine developmentSerological test and vaccine developmentSerological test and vaccine development

Identification of 106 antigensIdentification of 106 antigens

INFECTION AND IMMUNITY, July 2005, p. 4445–4450 Vol. 73Genome Scale Identification of Treponema pallidum Antigens Matthew McKevitt,1 Mary Beth Brinkman,1 Melanie McLoughlin,2 Carla Perez,1 Jerrilyn K. Howell,2 George M. Weinstock,4 Steven J. Norris,2,3and Timothy Palzkill1

progression in the breadth and intensity of humoral immunoreactivity throughout the course

of infection

Proteomics 2006, 6, 3294–3305Identification of candidate antigen in Whipple’s diseaseusing a serological proteomic approachMalgorzata Kowalczewska1, Florence Fenollar1, Daniel Lafitte2 and Didier Raoult1

Identification of 23 candidate proteins for Identification of 23 candidate proteins for serological diagnosis of Whipple’s disease serological diagnosis of Whipple’s disease

6 proteins common 6 proteins common to most patientsto most patientsto most patients to most patients

27


Reverse vaccinologyReverse vaccinology

•• Rational selection of antigenic epitopes Rational selection of antigenic epitopes completed by functional immunomicscompleted by functional immunomics

INFECTION AND IMMUNITY, Dec. 2002, p. 6817–6827 Vol. 70 Search for Potential Vaccine Candidate Open Reading Frames in the Bacillus anthracis Virulence Plasmid pXO1: In Silico and In Vitro ScreeningN. Ariel, A. Zvi, H. Grosfeld, O. Gat, Y. Inbar, B. Velan, S. Cohen, and A. Shafferman

11 putative targets11 putative targets

SCIENCE 10 MARCH 2000 VOL 287Identification of Vaccine Candidates Against Serogroup BMeningococcus by Whole-Genome SequencingPizza M et al.

Expression in Expression in E. coliE. coliof 350 putative antigensof 350 putative antigens

PerspectivesPerspectives

•• Extremely active field Extremely active field •• 3,963 ongoing bacterial genome projects3,963 ongoing bacterial genome projects•• Production of a greater range of diagnostic toolsProduction of a greater range of diagnostic tools•• Development of new antimicrobialsDevelopment of new antimicrobials•• Vaccine developmentVaccine development•• Ultimate bacterial phylogenyUltimate bacterial phylogeny

28

Greub – Culture Methods

Culture methods

Gilbert GREUBGilbert GREUB

Service of Infectious DiseasesUniversity Hospital Center

Institute of MicrobiologyUniversity of Lausanne

LausanneSwitzerland

Culture methods: importance1. Diagnostic:

‐ pathogenic agent ?

2. Discovery of new pathogens

3. Availability of strains:‐ Antibiotic susceptibility testing‐ antigen/antibody production (serology/immunohistochemistry)

‐ study the biology of a pathogen

Multiply only in cells

Remain undetected with axenic media routinely used in diagnostic laboratory

Intracellular bacteria

Facultative intracellular bacteria

Macrophage (ConA)Bacteria (Chlamydiales)

Strictintracellular bacteria

29


Growth on agar helps to:

Facultative Strict intracellular intracellular bacteria bacteria

101010...

Tneg10°10-1

10-2

10-3...

101010...

Tneg10°10-1

10-2

10-3...

Tneg10°10-1

10-2

10-3...

Obtain isolated colonies limiting dilutions‐ to screen for mutants‐ to avoid purification steps gradients,…

Cell culture challenges:

Identify strains based SDS‐page, MALDI‐TOF on phenotypes

Use selective mediaMycoplasma ?

Test the antibiotic …susceptibility

Etiological diagnosis = main challenge

Bl d l 2 bl d l 90% i i i

Endocarditis

Blood cultures: 2 blood cultures >= 90% sensitivity

Negative blood‐culture endocarditis‐ previous AB tt‐ fungal endocarditis‐ non‐infective endocarditis (marantic)‐ intracellular bacteria / fastidious organisms(Coxiella, Bartonella, T. whipplei, …)

Sensitivity Specificity

Histology 63% (62/98) 100% (118/118)PCR 61% (64/105) 100% (118/118)Valve cultures 13% (14/105) 98% (116/118)

Endocarditis

Valve cultures:

Valve cultures 13% (14/105) 98% (116/118)

Low sensitivity of culture Avoid culture if no suspicion of endocarditis …

Greub et al Am J Med 2005

30


Initially grown: ‐ in embryonated eggs (8‐10 days)

‐ in mice* animals shed C. burnetii‐ in guinea‐pigs* in urines/feces (biohazard)*diagnostic obtained by seroconversion in inoculated animals

Shell‐vial

Coxiella burnetii

‐ isolation of the Q fever agent from:‐ blood‐ valve‐ vascular prosthesis‐ bone marrow‐ vertebra/hip‐ …

‐ allows AB susceptibility testingRaoult, Vestris & Enea J Clin Microbiol 1990

Shell vial

Practically:‐ human embryonic lung fibroblasts (HEL)in MEM 10% fetal calf serum 1‐2% glutaminecontact‐inhibited growth allowing prolonged incubation

‐ 50’000 cells on cover slip

MEMCoverslip/HEL

Coxiella burnetii

50 000 cells on cover slipmonolayer obtained after 3 days at 37°C/CO2 5%

‐ 0.5 ml sample homogenized in PBS/0.5 ml MEMspinoculation (700 g 1h); then, washed with PBS

‐ cytopathic effect (enlarged cells), Gimenez staining, IF ‐ on day 6 or 10 (12/20 if negative): immunofluorescencesubcultures in flasks if > 70% of cells are infected

Raoult, Vestris & Enea J Clin Microbiol 1990Gouriet et al. J Clin Microbiol 2005

BartonellaTrench fever

Endocarditis

Cat‐scratch disease

31


Bartonella

Endothelial cells + centrifugationSensitivity 10x better than blood agar

p<0.001 from valvesp=0.045 from blood Fournier et al. J Clin Microbiol 2002

Cell culture >> axenic culture

Better sensitivity when:‐ Subculture blood cultures bottles on agar after 7 days‐ Congelation / saponin

Low CO2 production (undetected by automated systems)

BartonellaShell vial

Practically:‐ human endothelal cells (ECV304)in RPMI 15% fetal calf serum 2% glutamine

‐ read‐out: Gimenez staining, immunofluorescenceon day 15 (30/45 if negative)

‐ Sensitivity: serology >> PCR >> culture

Houpikian et al. Medicine 2005

Gouriet et al. J Clin Microbiol 2005

Tropheryma whippleiInitially, isolated using HEL cells…Genome analysis of amino‐acidPathways allowed to design a cell‐free culture medium

Renesto et al. Lancet 2003

32


Discovering new species

Microbiology: major role in the etiological diagnostis of infectious diseases

New agents remain to be discovered

20 h di d d i h l 3020 new pathogens discovered during the last 30 years(many agents of lower respiratory tract infection)

Culture‐based methods are open approachesbest suited to discover new pathogens:

‐ JNSP ‐ amoebal co‐culture

Legionella pneumophila

Mainly discovered during outbreaks

Chlamydia psittaci‐ outbreak in Uster in 1880‐ pandemia in 1929

psittacosis

Intracellular bacteria

Legionella pneumophilaFraser et al. 1977 New Engl J of Med

« Legionnaires’disease: description of an epidemic of pneumonia »

Lung Guinea pig Agar

• JNSP approach:

• « Je ne sais pas » i.e. « I do not know »

• Shell vial

– HEL and ECV304 cells

S i b Gi IF* f 21 d


– Screening by Gimenez + IF* after 21 days

– Identification by 16S rRNA PCR

• * patient serum

• 3861 samples: 175 bacterial species

32 contaminants

– Gouriet et al. J Clin Microbiol 2005

33


• JNSP axenic• only + JNSP

– Streptoccocus sp. (n=26) 7 (24%) 19

– Staphylococcus sp. (n=29) 2 (7%) 27


– Mycobacterium sp. (n=52) 6 (11.5%) 46

–– Francisella tularensis, Legionella pneumophila, Nocardia, Actinomyces, Chlamydia trachomatis, Tropheryma whipplei

Gouriet et al. J Clin Microbiol 2005

Environnement

Selection of Virulence traits

Amoebae as cell in a cell culture system• Selectively grows pathogens

Adaptation to macrophages

Lower respiratory tract

Adapted from: Greub et al. Clin Microb Rev 2004

Investigated sample

No lysis

Amoebal co‐culture

Photo Lyse/non lysées

serial dilutionsNo lysis

Lysis

34


1. Spinoculation2. Prevent ameobal encystment

‐ low temperatures (< 33°C)‐ humidified atmosphere

3. Use non‐nutritive medium to increase phagocytosis4 S l i i b l i

Amoebal co‐culture

4. Select permissive amoebal species5. Avoid antifungal agents6. Screening: PCR/IF/Gimenez7. Adapt protocols according to what you are looking for

‐ screening: Ziehl/auramine/…‐ pre‐treatment : acid/NaOH/…

Mycobacteria

Research program: Chlamydia‐related bacteria

Strains availability

STRAIN

« A pure culture is the foundation of all research on infectious diseases »Koch, 1881

Antigen for serology

Typing/taxonomyepidemiology

AntibioticCell biology

Antibodies forbacterial detection(ICH/direct IF/…) Animal models

Pathogenesis

susceptibilitytesting

GenomicProteomic

1

2 3 45

67 7

7

89

10 1112 12 12 13

7 7 1415 15

716

17 18

1919 20 21 21

2229

23

24 257

23

26 27

28

27

250 150

100

75

50

25

37

20

pH 3 pH 11

AC

B

D

LK

J IHG

E

O

MN

P

Q

R F

D

MW (kDa)

D

7

1

2 3 45

67 7

7

89

10 1112 12 13

7 1415 15

3716

17 18

19 20 21

2229

38

24 2536

23

26 2728

250 150

100

75

50

25

37

20

AC

B

D

LK

J IHG

E

O

M

N

P

Q

R F

T

MW (kDa)

U

35

S

35

1

2 3 45

67 7

7

89

10 1112 12 12 13

7 7 1415 15

716

17 18

1919 20 21 21

2229

23

24 257

23

26 27

28

27

250 150

100

75

50

25

37

20

pH 3 pH 11

AC

B

D

LK

J IHG

E

O

MN

P

Q

R F

D

MW (kDa)

D

7

1

2 3 45

67 7

7

89

10 1112 12 13

7 1415 15

3716

17 18

19 20 21

2229

38

24 2536

23

26 2728

250 150

100

75

50

25

37

20

AC

B

D

LK

J IHG

E

O

M

N

P

Q

R F

T

MW (kDa)

U

35

S

35

1

2 3 45

67 7

7

89

10 1112 12 12 13

7 7 1415 15

716

17 18

1919 20 21 21

2229

23

24 257

23

26 27

28

27

250 150

100

75

50

25

37

20

pH 3 pH 11

AC

B

D

LK

J IHG

E

O

MN

P

Q

R F

D

MW (kDa)

D

7

1

2 3 45

67 7

7

89

10 1112 12 13

7 1415 15

3716

17 18

19 20 21

2229

38

24 2536

23

26 2728

250 150

100

75

50

25

37

20

AC

B

D

LK

J IHG

E

O

M

N

P

Q

R F

T

MW (kDa)

U

35

S

35

35


Conclusions1. Diagnostic:

‐ low sensitivity /risk of contamination‐ biosafety issue‐ serology/molecular tests often better for diagnostic‐ only in trained laboratories

2 Discovery of new pathogens2. Discovery of new pathogens

3. Availability of strains:‐ AB susceptibility testing‐ typing (epidemiology)

‐ antigen/antibody production (serology/immunohistochemistry)

‐ study the biology of a pathogen

36

Rolain - Pharmacokinetics and antibiotic dosage

UMR 6236

Educational Workshop

« Infections by intracellular pathogens : from the laboratory to the clinic »

Ph ki ti d tibi ti dPharmacokinetics and antibiotic dosage« The Q fever experience »

Pr Jean-Marc ROLAIN

Pôle des Maladies Infectieuses et URMITE – CNRS UMR 6236 – IRD 198 –Université de la Méditerranée – Marseille – France

[email protected]

20th ECCMID – VIENNA – APRIL 2010

Target cells and subcellular localisation of intracellular bacteria

Obligate intracellular bacteria Target cells Subcellular

localisation

Rickettsia Endothelial cells Cytosol

UR

Ehrlichia

Coxiella burnetii

Tropheryma whipplei

Macrophages, PMN

Macrophages

Macrophages

Phagosome

Phagolysosome

Phagolysosome

pH 6,5

Bacteria

Early phagosome

Destruction of bacteria in phagocytic cellsDestruction of bacteria in phagocytic cellsStrategy for survival of intracellular bacteria in cellsStrategy for survival of intracellular bacteria in cells

Rickettsia spp.Shigella flexneri

Acidification

Late phagosome

Lysosomes

cytosol

nucleus

Phagolysosome(complete fusion)

Killing of the bacteria

pH 4,5

Acidification

Late phagosome(incomplete fusion)

Phagolysosome(complete fusion)

pH 4,5

BartonellaBrucellaLegionellaSalmonellaChlamydiaYersiniaEhrlichia

Coxiella burnetiiTropheryma whippleiFrancisella tularensisMycobacterium tuberculosis

37


Extracellular medium Phagolysosome

Active drug

Inactive drug

cytosoldiffusion

bacteria

pH 4.5

pinocytosis

cytosolic activity

subcellular distribution

subcellular activity

metabolism andinactivation

Antibiotic uptakeAntibiotic uptake

Antibiotic Mode of entry Cytosol Lysosomes PH of optimum activity

Aminoglycosides

Betalactams

Chloramphenicol

Pinocytosis

Diffusion

Diffusion

+

++

+++

Unknow

7

7

7

Antibiotic uptake, subcellular localisation and pH of optimum activity of antibiotics

Erythromycin

Fluoroquinolones

Rifampin

Tetracyclines

Transport

Unknow

Diffusion

Diffusion

+

++

++

++

+++

++

++

Unknow

7.8

8

<5

6.6

Symbols : + = low concentration ; ++ = medium concentration ; +++ = high concentration

Coxiella burnetii :Coxiella burnetii : BacteriologyBacteriologyRickettsia rickettsiiRickettsia prowazekiiOrientia tsutsugamushiEhrlichia chaffeensisEhrlichia canisCowdria ruminantumEhrlichia phagocytophilaAnaplasma marginaleWolbachia pipientisEhrlichia sennetsuNeorickettsia helminthoeca

Bartonella quintanaBartonella henselae

α1PROTEOBAC

Tribe Species Genus

Rickettsia rickettsiiRickettsia prowazekii Rickettsia

Rickettsieae Rickettsia tsutsugamushiRochalimea quintana RochalimeaCoxiella burnetii Coxiella

Ehrlichia canisEhrlichia phagocytophila Ehrlichia

Pleomorphic CoccobacillusGram-negative

0.2 - 0.7 mm Gimenez stainingAgent of Q fever

Bartonella henselaeBartonella talpaeBartonella bacilliformisBrucella melitensis

Coxiella burnetiiRickettsiella grylliLegionella pneumophilaWolbachia persicaFrancisella tularensis

Eperythrozoon ovisHemobartonella felisMycoplasma pneumoniaeUreaplasma urealyticumChlamydia trachomatis

α2

γ

A B

CTERIA

Grampositive

Ehrlichia phagocytophila EhrlichiaEhrlichiae Ehrlichia sennetsu

Cowdria ruminantium CowdriaNeorickettsia helminthoeca Neorickettsia

Bartonellaceae Bartonella bacilliformis BartonellaGrahamella talpae Grahamella

Wolbachia pipientis WolbachiaWolbachieae Wolbachia persica

Rickettsiella grylli Rickettsiella

Anaplasmamarginale AnaplasmaAnaplasmataceae Eperythrozoon ovis Eperythrozoon

Hemobartonella felis Hemobartonella

38


Coxiella burnetii : Coxiella burnetii : BacteriologyBacteriology

Obligate intracellular bacteriumTarget monocytes/macrophagesAny animalsAmoebaMultiplication within theMultiplication within the phagolysosome of macrophagesSurvives in acidic vacuole

(low pH activates metabolism)

Coxiella burnetii :Coxiella burnetii : BacteriologyBacteriology

Spore-like formExtremely resistantSurvival under harsh conditions :– 60 min at 600 C60 min at 60 C– 10 months at 200 C– in formalin 0.5 %– UV-irradiation

Clinical presentation %

Isolated fever 14Hepatitis 40Pneumonia 17P i + H titi 20

Acute Q fever in 1,070 patients*Acute Q fever in 1,070 patients*

Pneumonia + Hepatitis 20CSF sampling 4 Meningitis 0,5 Meningoencephalitis 1Pericarditis 1Myocarditis 1Not determined

*Patients classified in 1 category only

3

Raoult D., et al. Q fever 1985-1998. Clinical and epidemiologic features of 1,383 infections.

Medicine (Baltimore). 2000;79:109-23.)

39


Chronic Q feverChronic Q feverPrevalence of various formsPrevalence of various forms

N° of identified cases(n=313) %

Endocarditis 229 73Vascular infection 25 8Pregnancy (mothers and babies) 20 6Pregnancy (mothers and babies) 20 6Chronic hepatitis 8 3Osteoarticular infection 7 2Chronic pericarditis 3 1Adenopathies 1 <1Splenic pseudotumor 1 <1Lung pseudotumor 1 <1Chronic neuropathy 1 <1No identified foci 6 2

Chronic Q feverChronic Q feverFactors influencing the evolution to persistent or chronic Q fever

Host factorsHost factorsImmunosuppressionPatients with immunosupression (cancers, lymphomas, or HIV infection)persistent infection in athymic mice, reactivation of infection with steroids orwhole body irradiation in mice and guinea pigs, endocarditis in mice receivingcyclophosphamidecyclophosphamide

ValvulopathyHuman endocarditis and previous valvulopathy endocarditis in guinea pigs withdamaged cardiac valves

PregnancyEndocarditis in pregnant mice chronic Q fever in pregnant mammals

Primary infectionAsymptomatic (60%)

Symptomatic

Exposure

- aerosol- milk product

Chronic Infection

Pathophysiology: acute Q feverPathophysiology: acute Q fever

mild undiagnosed

severe, diagnosed

(3-7 %), males, middle age

feverpneumonia

hepatitis

During pregnancy, abortion, chronic carriage

Valve lesion (2 %), valve abnormality

Endocarditis (30-60%) in 2 years

Chronic Infection in Special Hosts

Raoult D, et al. Natural history and pathophysiology of Q fever. Lancet Infect Dis. 2005 ;5:219-26

Cancer (lymphoma)

Endocarditis

40


DiagnosisDiagnosis

Serology (MIF)Isolation (Shell vial technique): blood and heart valvesImmunofluorescence or Immunohistochemistry: heart valvesPCR and real-time PCR

• The survival and multiplication of C burnetii in an acidic vacuole prevents antibiotics from killing the bacteria

• Increasing pH with lysosomotropic agents such as chloroquine restores the bactericidal activity of doxycycline.

TreatmentTreatment

10004,8 5,3 5,7 6,8

Control Amantadine 1µg/ml Chloroquine 1µg/ml NH4Cl 1 mg/ml

pH

Based on these data, a treatment regimen combining doxycycline and chloroquine was proposed and demonstrated to be effective in treating people with chronic infection

0,1

1

10

100

% RVB

Rifampin Pefloxacin Doxycycline

Treatment : acute Q feverTreatment : acute Q feverUsually acute Q fever resolves without treatment within 15 daysVarious antibiotics have been reported to be effective :– Ofloxacin, Pefloxacin– Erythromycin– Chloramphenicol– Cotrimoxazole

C ft i– CeftriaxoneDoxycycline (200 mg/day) for 2-3 weeks remains the antibiotic-regimen of choiceQuinolones should be considered in Q fever meningoencephalitis

Raoult D. Antimicrob Agents Chemother 1993; 37:1733-6.

41


Treatment Treatment Antibiotic regimens for Q fever endocarditis Antibiotic regimens for Q fever endocarditis

Duration

(months)

Succes

(%)

Relapse

(%)

Mortality

(%)

Doxycycline Lifetime 0 100 50

Raoult D, et al. Treatment of Q fever endocarditis: comparison of 2 regimens containing doxycycline and ofloxacin or hydroxychloroquine. Arch Intern Med. 1999 ;159:167-73.

Doxycycline +

quinolone

> 36 < 30 > 50 < 5

Doxycycline +

OH-chloroquine

> 18 > 80 < 5 < 5

Treatment : in vivo dataTreatment : in vivo data

Relapse rate < 5% if treatment > 18 monthsmonitoring OHCQ to obtain 1 +/- 0.2 µg/mlDoxycycline : 200 mg/daycompliance (photosensibilisation)Monitoring of Phase I antibodies (decrease)Monitoring of Phase I antibodies (decrease)

However : heterogeneity of biological response between patients Duration treatment range from 18 to 36 months

Doxycycline level?Doxycycline MIC of strains?Resistant strains?

First step

J Infect Dis 2003, 188 : 1322-1325

42


R2 = 0,44

6

7

8

9

10

11

12

13

14

e (µ

g/m

l) af

ter 1

yea

r

Evolution of doxycycline level in sera during treatment : n = 24 patientsDoxycycline 200 mg/day

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9

Doxycycline (µg/ml) after 3 months

Dox

ycyc

line

Serum doxycycline level at 1 year > 3 months ; p < 0.05Marked interpatients variation : from 0.06 to 12.92 µg/ml!!

3 14

5,29

4

5

6

7

ne (µ

g/m

l)

Correlation between doxycycline level and decrease of Phase I antibodies

3,14

0

1

2

3

< 2 dilutions > 2 dilutions

Dox

ycyc

lin

p = 0,003*

Consequence : adapt doxycycline posology in order to obtain 5 µg/ml

Cured

56789

1011121314

xycy

clin

e (µ

g/m

l)

2400

3200

4000

4800

5600

6400

IgG

1 tit

er

012345

Time (Days)

Dox

0

800

1600

2400

Doxycycline 1,16 8,5 12,85 12,92

IgG1 6400 3200 800 400

0 142 331 436

43


Not cured

56789

1011121314

xycy

clin

e (µ

g/m

l)

2400

3200

4000

4800

5600

6400

IgG

1 tit

er

012345

Time (Days)

Dox

0

800

1600

Doxycycline 0 4,07 2,5 3,5

IgG1 3200 3200 3200 3200

0 118 279 384

Doxycycline MIC?

Second step

2

3

4

NA

copi

es (l

og)

Activity of Doxycycline - MIC detection by using Q PCR (Q212)

1µg/ml

0.5µg/ml

Negative control

0

1

0 2 4 6 8 10 12

DN

Time (days)

2µg/ml4µg/ml

8µg/ml

Q212 reference strain - MIC = 2 µg/ml

44


Annals New York Acad Sci 2005 Dec;1063:252-6.

Third step

• 13 new isolates of Coxiella burnetii using a real-time quantitative PCR assay

• MICs against doxycycline ranged from 1 to 8 µg/mL

• telithromycin from 0.5 to 2 µg/mL

• Doxycycline-resistant strains exist either in humans or animals (3 strains)

JM Rolain et al. Annals New York Acad Sci 2005 Dec;1063:252-6.

Fourth step

45


• 16 patients with C. burnetii endocarditis• Retrospective analysis

6 1

Fast serologic response = decrease

9

16 Patients

Favorable clinical response Therapeutic failure

115

J.M. Rolain, A. Boulos, M.N. Mallet, and D. Raoult*. Antimicrobial Agents and Chemotherapy 2005

Low serological response

0.5-1

4.57+2.3

3.0+0.8

P<0.050.4

Death during the course of the treatment

8

3.5

MIC doxycycline

Ratio

Doxycycline (serum)Mean at 1 year

>1

2.33+1

4.6+1.6

pof Phase I antibodies > 2 dilutions in 1 year

Fast serological response

Patient (Germany) MIC = 8µg/ml

456789

101112

xycy

clin

e (µ

g/m

l)

25600

51200

hase

I an

tibod

y tit

er

0123

Time (Days)

Dox

0

IgG

Ph

Doxycycline (plasma) IgG1 (serum)

D o xycycline ( p lasma) 0 4 ,4 1 3 ,6 1 4 ,73 3 ,0 8 2 ,19 5,5 5,0 1

Ig G1 ( serum) 2 56 0 0 2 56 0 0 2 56 0 0 2 56 0 0 2 56 0 0 2 56 0 0 2 56 0 0 2 56 0 0

13 2 5 56 6 4 111 151 2 3 9 3 54

Doxycycline = 3.57 [2.32 - 4.82] µg/ml

Increase posology of doxycycline if < 5 µg/ml to obtain biological response

Last step

46


Patient with a rapid serological response at 1 year of treatment

Patient with a slow serological response for which doxycycline was increased to 300 mg/day

Treatment Treatment -- SummarySummary

Treatment of chronic forms : at least 18 months

Doxycycline should be monitored and ≥ 5µg/ml serum – If not, increase the posology to 300-500 mg/day

OH-Chloroquine should be 1 ± 0.2 mcg/ml

When possible, the strains susceptibility should be tested to adapt doxycycline posology

Resistance is increasing (MIC > 2 µg/ml)

47

Documents

GIKAS ESCAR-2010final.ppt [Kompatibilitätsmodus]