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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
Gikas - Evolution of serology
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
Gikas - Evolution of serology
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 (-)
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 1/600 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
Gikas - Evolution of serology
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
Gikas - Evolution of serology
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
Gikas - Evolution of serology
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
Gikas - Evolution of serology
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
Raoult D, Parola P, eds. Rickettsial diseases. Informa healthcare 2008
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.
Raoult D, Parola P, eds. Rickettsial diseases. Informa healthcare 2008
9
Gikas - Evolution of serology
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
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 (-)
10
Gikas - Evolution of serology
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
Raoult D, Parola P, eds. Rickettsial diseases. Informa healthcare 2008
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
Gikas - Evolution of serology
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
Raoult D, Parola P, eds. Rickettsial diseases. Informa healthcare 2008
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
Gikas - Evolution of serology
Automation of serodiagnosis with the Multiplexed automated corpuscular antigenic microarray (MACAM)
Coxiella burnetii, Chlamydophila pneumoniae, Chlamydia psittaci, Legionella pneumophila, Francisella tularensis, Mycoplasma pneumoniae
Gouriet F, et al. Clin Microbiol Infect; 2008
Multiplexed automated corpuscular antigenic microarray (MACAM)
Algorithm of interpretation for blood negative-culture endocarditis
Gouriet F, et al. Clin Microbiol Infect; 2008
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
Gikas - Evolution of serology
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.
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
Fournier - Genomics and clinical application
>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
Fournier - Genomics and clinical application
Two objectivesTwo objectives
Detection Detection -- IdentificationIdentification
Two objectivesTwo objectives
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
Fournier - Genomics and clinical application
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
Fournier - Genomics and clinical application
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
Fournier - Genomics and clinical application
Two objectivesTwo objectives
Detection Detection -- IdentificationIdentification
Two objectivesTwo objectives
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
Fournier - Genomics and clinical application
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
Fournier - Genomics and clinical application
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
Fournier - Genomics and clinical application
• 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
Fournier - Genomics and clinical application
Creation of the free-access, online MST-Rick database(http://ifr48.timone.univ-mrs.fr/MST_BHenselae/mst).
Phenotype predictionPhenotype prediction
Culture medium designCulture medium design
Phenotype predictionPhenotype prediction
Antibiotic resistanceAntibiotic resistance Detection of virulenceDetection of virulence
Source of information Source of information for metabolismfor metabolism
• Several essential aminoacid pathways are lacking
24
Fournier - Genomics and clinical application
Folate Folate biosynthesis biosynthesis and enzymesand enzymes
Dihydropteroate synthase y p y(DHPS)
Sulfamethoxazole target
Dihydrofolate reductase (DHFR)
Trimethoprim target
Phenotype predictionPhenotype prediction
Culture medium designCulture medium design
Phenotype predictionPhenotype prediction
Antibiotic resistanceAntibiotic resistance Detection of virulenceDetection of virulence
25
Fournier - Genomics and clinical application
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
Phenotype predictionPhenotype prediction
Culture medium designCulture medium design
Phenotype predictionPhenotype prediction
Antibiotic resistanceAntibiotic resistance Detection of virulenceDetection of virulence
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
Fournier - Genomics and clinical application
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
Fournier - Genomics and clinical application
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
Greub – Culture Methods
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
Greub – Culture Methods
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
Greub – Culture Methods
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
Greub – Culture Methods
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
Discovering new species
– 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
Greub – Culture Methods
• JNSP axenic• only + JNSP
– Streptoccocus sp. (n=26) 7 (24%) 19
– Staphylococcus sp. (n=29) 2 (7%) 27
Discovering new species
– 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
Greub – Culture Methods
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
Greub – Culture Methods
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
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
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
Rolain - Pharmacokinetics and antibiotic dosage
• 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
Rolain - Pharmacokinetics and antibiotic dosage
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