DU de thérapeutiques anti-infectieuses. Coxiella burnetii. Pierre-Edouard Fournier. Grenoble, 19 janvier 2018

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1 DU de thérapeutiques anti-infectieuses Coxiella burnetii Pierre-Edouard Fournier Grenoble, 19 janvier 2018

Q fever = Query fever «Then the suspicion arose and gradually grew into a conviction that we were dealing with a type of fever which had not previously been described.

2 Q fever = Query fever «Then the suspicion arose and gradually grew into a conviction that we were dealing with a type of fever which had not previously been described. It became necessary to give it a name, and «Q fever» was chosen to denote it until fuller knowledge should allow a better name.» Edward H. Derrick Brisbane, Queensland, Australie, 1937

3 Q fever Caused by Coxiella burnetii Strictly intracellular Gram-negative bacterium Zoonotic reservoir Infects endothelial cells and macrophages Antigenic phase variation (LPS truncated in phase II)

4 Coxiella burnetii the ultimate survivor Multiplies at acidic ph (ph =4) Spore-like form C: 7-10 months Refrigerated (4 C) meat: > 1 month Milk at room temp.: > 40 months Highly resistant in the environment

5 Coxiella burnetii

Phase 1 Infectious Antigenic phase variation Phase 2 Not infectious Resistant to macrophages Only form found in animals Slow multiplication in cells Responsible for a late and massive antibody answer

6 Phase 1 Infectious Antigenic phase variation Phase 2 Not infectious Resistant to macrophages Only form found in animals Slow multiplication in cells Responsible for a late and massive antibody answer in chronic Q fever Destroyed by macrophages Absent in nature, obtained after culture (cell culture, embryonated eggs) Rapid multiplication in cells Responsible for an early antibody response in acute Q fever 17-kb deletion in phase 1

7 Epidemiology of Q fever Reservoirs Sheep Goats Cattle Cats Dogs Birds (pigeons) Humans Contaminating factors Uterus Placenta Stool Urine Milk Manure Ticks (140 species) Sperm Blood Powders (bioterrorism)

8 A dangerous human pathogen Infectious dose < 10 bacteria Class III biohazard agent CDC category B bioterrorism agent (Amano & Williams. J Bacteriol 1984; 160: )

9 Transmission to humans Inhalation of contaminated aerosols +++ Ingestion of raw milk products Tick-bite Trans-placental transmission Inter-human transmission (autopsy, delivery, transfusion) Sexual transmission?

10 Worldwide distribution (except New Zealand?) 7 th most frequent zoonosis in Europe (~860 cases/y) Epidemiology of Q fever Not a reportable disease in most countries => true prevalence? Increased prevalence around breeding areas

11 Genotypes, geotypes or pathotypes? 11

12 Physiopathology of Q fever Contamination Primo-infection Asymptomatic (60%) - aerosols ++ - Milk products Pregnancy: delivery, chronic infection Valvular defect Acute Q fever Chronic Q fever At-risk patients Cancer (lymphoma) Mild, flu-like symptoms Symptomatic (40%) Severe (3-7 %) Isolated fever Pneumonia Hepatitis Endocarditis (40 %) within 2 years Endocarditis

Acute Q fever, a polymorphic disease Isolated fever

manifestations: adenopathies, pancreatitis, haemolytic

13 Acute Q fever, a polymorphic disease Isolated fever (14%) Atypical pneumonia (17%) Hepatitis (40%) Méningo-encéphalitis (1%) Pericarditis (1%) Myocarditis (1%) Chronic fatigue (5-10%) Other manifestations: adenopathies, pancreatitis, haemolytic anemia, diarrhoea, spleen rupture... Mortality < 0.5% (Raoult et al. Medicine; 2000)

14 Acute Q fever Variations from country to country Hepatitis Pneumonia Febrile illness Basque county Canada Guiana Netherlands Switzerland Andalusia Australia France

15 Q fever in children Few cases despite a frequent exposure Outbreak in Switzerland: 100% 90% 80% 70% 60% 50% 40% 30% <14 ans = 19% 20% seropositivity but 10% only 5% symptomatic 0% (Maltezou et al. Am J Trop Med Hyg; 2004) < 14 ans > 14 ans Children are less symptomatic than adults Asymptomatic Symptomatic

16 Q fever in Guiana (Edouard et al. Am J Trop Med Hyg. 2014;90:915-9; Epelboin et al. Clin.Infect.Dis. 2012;55:67-74) Q fever outbreak in French Guiana since cases/100,000 inhabitants in 2005 (Grangier et al. Bull.Veille.Sanitaire.2009;10:2-4) Higher antibody response Cayenne Metropolitan France Acute Q fever 17.5/100, /100,000, P<10-2 Pneumonia 83%, 24% of CAP 8%, P<10-2 Hepatitis 32% 54%, P<10-2 Endocarditis 7% 17%, P = 0.017

burnetii strain 1,00E+04 Blood BALB/c 1,00E+08 1,00E+07 Blood SCID 1,00E+03 1,00E+06 1,00E+05 1,00E+02 1,00E+01 Guyane

17 DNA copies log (10) DNA copies log 10 Strain 175 (Mahamat et al. Emerg.Infect.Dis. 2013;19:1102-4) Reservoir = three-toed sloth (Davoust et al. Emerg.Infect.Dis. 2014;20:1760-1) Most virulent C. burnetii strain 1,00E+04 Blood BALB/c 1,00E+08 1,00E+07 Blood SCID 1,00E+03 1,00E+06 1,00E+05 1,00E+02 1,00E+01 Guyane NMI 1,00E+04 1,00E+03 1,00E+02 Guyane NMI 1,00E+00 D3 D7 D14 D28 Time post-infection (days) 1,00E+01 1,00E+00 D3 D7 D14 D28 Time post-infection (days) Specific genotype

18 Chronic Q fever Blood culture negative endocarditis Vascular infection Pregnancy Chronic hepatitis Osteo-articular infection Chronic pericarditis Adenopathies Pseudo-tumor of the spleen Pseudo-tumor of the lung Chronic neuropathy Unidentified location N of identified cases (n=313) % <1 1 <1 1 <1 1 6 <1 2

19 Q fever endocarditis P Diagnostic delay (months) 18 5,7 <0.01 Cardiac Insufficiency 9 3 <0.05 Hepatomegaly 12 5 <0.01 Splenomegaly 7 4 Elevated liver enzymes 7 3 Thrombocytopenia 7 6 VS > Fever Valvular replacement 7 10 Death within 3 years following the diagnosis 6 0 <0.01

20 Q fever and pregnancy Natural outcome Abortion or stillbirth : 38% Prematurity, small birthweight : 33% Relapse : 4%

21 number of serum screened number of positive screening Annual number of patients tested for Q fever in our laboratory number of serum screened and number of positive screening year number of screened serum number of positive screening : sera analyzed, positive on screening (30%) Increasing Q fever serology queries over years (except in 2006 and 2007) Increasing numbers of positive sera on screening (two peaks: 1992 and 2004)

22 number of Q fever Numbers of diagnosed acute and chronic Q fever cases Chronic Q fever: : stable incidence ~33/year Since 2006: Important increase ~246/year Acute Q fever: Regular increase year chronic Q fever acute Q fever

23 Peaks in 1993 (181 cases), 1999 (268) Changing epidemiology of Q fever in Germany, Hellenbrand W, Emerg Infect Dis Sep- Oct;7(5): USA: In other countries 436 cases between 1978 and 1999 Reportable since % increase from 2000 to 2004 National surveillance and the epidemiology of human Q fever in the United States, McQuiston JH, Am J Trop Med Hyg Jul;75(1): Germany: Few cases diagnosed in East Germany (except Thuringia outbreak in ) Irregular cyclic variation 4-6-year intervals between peaks Max in 1964: 437 cases

24 number of acute Q fever Seasonality of acute Q fever Marseille area Annual peaks between April and September Minimum beween October 10 and January Jan-00 May-00 Sep-00 Jan-01 May-01 Sep-01 Jan-02 May-02 Sep-02 Jan-03 May-03 Sep-03 Jan-04 May-04 Sep-04 Jan-05 May-05 month Sep-05 Jan-06 May-06 Sep-06 Jan-07 May-07 Sep-07 Jan-08 May-08 Sep-08 Jan-09 May-09 Sep-09 acute Q fever

25 Q fever in Southern France 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0,05 0 Prevalence of acute Q fever between 1990 and 2016 in the Bouches du Rhône area (prevalence for inhabitants) Martigues Aix en Provence Marseille

26 Annual distribution of 109 Q fever cases diagnosed between 1990 and 1996 in Martigues Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

27 Why Martigues? Martigues, : Prevalence 10 times greater than Marseille (30 km) Flat landscape Arles La Crau Aix en Provence Town exposed to NW wind (Mistral) blowing over La Crau Martigues Mediterranean sea Marseille La Crau : breeding area for over sheep

28 Mistral in Martigues, 1998 Oct Nov Dec Jan Feb Mar Apr Sep May Lambing Mistral > 8m/s Cases of Q fever 2 months later Aug Jun Tissot-Dupont et al. Emerg Infect Dis; 2004 Jul

29 number of Q fever Q fever may cause outbreaks Q fever outbreaks in France Chamonix* Cholet* Banon* Briançon* year chronic Q fever acute Q fever Acute and Chronic Q fever from 1985 to * places where outbreaks were reported.

30 Outbreaks in France Banon: cases patients and personnel in a psychiatric hospital close to a farm with goats A cluster of Coxiella burnetii infections associated with exposure to vaccinated goats and their unpasteurized dairy products. Fishbein DB, Am J Trop Med Hyg Jul;47(1): Briançon: cases Contamination by aerosols from a slaughterhouse Investigation of a slaughterhouse-related outbreak of Q fever in the French Alps. Carrieri MP, Eur J Clin Microbiol Infect Dis Jan;21(1):17-21.

31 Outbreaks in France Chamonix: cases No identified source Role of sex, age, previous valve lesion, and pregnancy in the clinical expression and outcome of Q fever after a large outbreak. Tissot-Dupont H, Clin Infect Dis Jan 15;44(2): Epub 2006 Dec 6. Cholet: cases In majority employees from a meat transformation factory Unpublished data

Outbreaks in Europe 1987-2009 25, 26 39 40 27 28 unp

32 Outbreaks in Europe , unp 29, 30 c, 31 b d,14 i h,j f,m,41 a n l e e g 35 36

33 Outbreak in the Netherlands Reportable in humans since : ~17 cases per year 2007: 192 cases (pneumonia) 2008: 1,000 cases 2009: 2,300 cases 2010: > 4,000 cases Southern part of the country 350,000 goats, 1.1 M sheep, 4 M bovines CDC Health Advisory, May 12, 2010

34 Diagnosis of Q fever Elevated liver transaminases (85%) Thrombocytopenia (25%) Serology using indirect immunofluorescence Enables estimation of the evolutive stage of the disease Determination of IgG, M, A to phases 1 and 2

35 Diagnosis of Q fever Acute Q fever: IgG II > 1:200 and IgM II > 1:50 Chronic Q fever: IgGI > 1:800 PCR from any specimen targeting the is1111 repeat (19-55 copies per strain) Cell culture (endothelial cells) restricted to BSL3 laboratories

36 Axenic culture of C. burnetii: what a challenge! (Omsland A et al. Proc Natl Acad Sci U S A. 2003;106:4430-4; Singh S et al. J Clin Microbiol. 2013;51: ) Genome analysis Prediction of the main metabolic pathways Identification of the growth requirements Design of an axenic medium Acidified cystein citrate medium 2 (ACCM2)

37 Positive culture on ACCM2 agar at day 7

38 Coxiella burnetii and antibiotics

39 Susceptibility to antibiotics C. burnetii is naturally resistant to -lactams, aminoglycosides and chloramphenicol Susceptible to tetracyclines, cotrimoxazole, fluoroquinolones, rifampicin Inconstant susceptibility to macrolides (Rolain et al. Antimicrob Agents Chemother; 2001)

40 Treatment of acute Q fever In most case, self-limiting disease in 15 days Reference therapy = doxycycline, 200 mg/j, for 2-3 weeks In patients with meningo-encephalitis: fluoroquinolone In case of auto-immune manifestation, short corticosteroid course (Raoult. Antimicrob Agents Chemother; 1993) In patients with identified valvular defect: Doxycycline + hydroxy-chloroquine, 12 months

41 Treatment of chronic Q fever In vitro studies: Phagolysosomes from C. burnetii-infected cells maintain an acidic ph during infection Alkalinisation of the phagolysosome => critical to obtain pour obtenir a bactericidal effect of antibiotics (Maurin et al. J Infect Dis; 1992, Raoult et al. Arch intern Med; 1999) Impact on the treatment

42 Treatment of chronic Q fever Coxiella burnetii + P388 D1 Doxycycline ph 4.8 Bacteriostatic Doxycycline + OHchloroquine (1 mg/ml) ph 5.7 Bactericidal Rifampicin Pefloxacin Doxycycline (Maurin et al. J Infect Dis; 1992)

43 Treatment of chronic Q fever Compared efficiency of various protocols Duration (months) Success (%) Relapse (%) Doxycycline > Mortality (%) Doxycycline + quinolone Doxycycline + hydroxychloroquine > 36 < 30 > 50 > 18 > 80 < 15

44 Treatment of chronic Q fever Doxycycline + OH-chloroquine 18 to 36 months. Adapt doxycyline posology to MIC (> 5 μg/ml) and check OHchloroquine serum level (< 1 μg/ml) Or doxycycline + fluoroquinolone 36 à 60 months Treatment duration dependent on the serological evolution (stop when IgG I < 1:800) Pregnancy: cotrimoxazole until 2 weeks before delivery In case of immunosuppression, doxycycline for life

45 Antibiotic resistance in Coxiella burnetii? 2010 In vitro selection gyra Asp87Gly gyrb Ser431Pro and Met518Ile parc Asp69Asn, Thr80Ile, and Gly104Ser

46 Evaluation of antibiotic susceptibility Cell culture Axenic culture on ACCM2 agar + Etest (Clay et al. Int J Antimicrob Agents. 2017)

47 Summary of American CDC Q Fever Treatment recommendations Treatment of chronic Q fever should be initiated only after diagnostic confirmation. Doxycycline is the drug of choice, and 2 weeks of treatment is recommended for adults, children aged 8 years, and for severe infections in patients of any age. Children aged <8 years with uncomplicated illness may be treated with trimethoprim/sulfamethoxazole or a shorter duration (5 days) of doxycycline. Women who are pregnant when acute Q fever is diagnosed should be treated with trimethoprim/sulfamethoxazole throughout the duration of pregnancy. Serologic monitoring is recommended after an acute Q fever infection to assess possible progression to chronic infection.

48 Vaccination? Available in Australia only Highly exposed populations

Q Fever What men and women on the land need to know

Q Fever What men and women on the land need to know Dr. Stephen Graves Director, Australian Rickettsial Reference Laboratory Director, Division of Microbiology, Pathology North (Hunter) NSW Health Pathology,