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Coxiella burnetii-positive PCR in febrile patients in
rural and urban Africa
Emmanouil Angelakis, Oleg Mediannikov, Cristina Socolovschi, Nadjet
Mouffok, Hubert Bassene, Adama Tall, Hamidou Niangaly, Ogobara Doumbo,
Abir Znazen, Mhammed Sarih, et al.
To cite this version:
Emmanouil Angelakis, Oleg Mediannikov, Cristina Socolovschi, Nadjet Mouffok, Hubert Bassene, et
al.. Coxiella burnetii-positive PCR in febrile patients in rural and urban Africa. International Journal
of Infectious Diseases, Elsevier, 2014, 28, pp.107 - 110. �10.1016/j.ijid.2014.05.029�. �hal-01774657�
Coxiella
burnetii-positive
PCR
in
febrile
patients
in
rural
and
urban
Africa
Emmanouil
Angelakis
a,*
,
Oleg
Mediannikov
a,b,
Cristina
Socolovschi
a,
Nadjet
Mouffok
c,
Hubert
Bassene
a,b,
Adama
Tall
d,
Hamidou
Niangaly
e,
Ogobara
Doumbo
e,
Abir
Znazen
f,
Mhammed
Sarih
g,
Cheikh
Sokhna
a,b,
Didier
Raoult
a,ba
URMITE,UM63,CNRS7278,IRD198,Inserm1095,13005Marseille,France
b
CampusCommunUCAD-IRDofHann,BP1386,CP18524,Dakar,Senegal
c
ServicedesMaladiesInfectieuses,CentreHospitalo-Universitaired’Oran,Oran,Algeria
d
InstitutPasteurdeDakar,Dakar,Senegal
e
MalariaResearchandTrainingCentre,FacultyofMedicine,UniversityofSciencesTechniquesandTechnology,Bamako,Bamako,Mali
f
LaboratoryofMicrobiologyandLaboratoryofResearch‘MPH’,HabibBourguibaUniversityHospital,Sfax,Tunisia
gLaboratoiredesMaladiesVectorielles,InstitutPasteurduMaroc,Casablanca,Morocco
1. Introduction
Feverisacommon problemforwhichpatients seekmedical advice.Investigationsofthespectrumofetiologyofunknownfever inAfricahaveprogressedrapidlyduringrecentyears,resultingin an improvementin knowledge aboutbacterial infection.1 Most
uninvestigatedcausesofdeathinSub-SaharanAfricaaretheresult ofinfectiousdiseases.1Moreover,travelersinAfricaareexposedto
various health risksin unfamiliar environments, and fever is a commonproblemintravelers.2–4Mostcasesareacquiredin
Sub-Saharan Africa, and spotted fever group (SFG)rickettsioses are
secondonlytomalariaasthemostcommonlydiagnoseddiseases inreturneeswithasystemicfebrileillness.2,3In2008,westartedto
investigatethecausesoffeverofunknownorigin(FUO)inAfrica, andparticularlyinSenegal,toidentifyappropriatestrategiesfor casemanagementinruralcommunities.5–8
Qfeverisaworldwidezoonosiswithmanyacuteandchronic manifestationsthatis causedbythepathogenCoxiellaburnetii.9 Clinical findings in Q fever infection are often confusing, and primaryinfectionisasymptomaticinapproximately60%ofcases.9
InfectionswithC.burnetiioccurthroughoutAfrica.10Insomecases
ofQfever,bacteriamaypersistforyearsdespiteantibiotictherapy, andithasbeenproposedthatinendemicregions,humansmay excreteC.burnetii,evenwithoutevidentclinicalsigns,foralong time.6 A high prevalence of Q fever wasidentified recently in several villages in Senegal, indicating that Q fever should be
ARTICLE INFO Articlehistory: Received14April2014
Receivedinrevisedform27May2014 Accepted30May2014
CorrespondingEditor:EskildPetersen, Aarhus,Denmark Keywords: Qfever Africa Coxiellaburnetii Genotype
Multi-spacersequencetyping
SUMMARY
Objectives:QfeverhasbeenreportedthroughouttheAfricancontinent.Theobjectiveofthisstudywasto detectthepresenceofCoxiellaburnetiiinfebrilepatientsfromAfrica.
Methods:Bloodsamplesfromfebrileandnon-febrilepatientsfromsixAfricancountriesandfromFrance wereinvestigatedretrospectivelyforQfeverinfectionbymolecularassaystargetingtheIS1111and IS30Aspacers.
Results:Wetested1888febrilepatientsfromSenegal,Mali,Tunisia,Algeria,Gabon,andMoroccoand foundonemaleadultpatient(0.3%)infectedwithC.burnetiiinAlgeriaandsixpositivepatients(0.5%)in Senegal.ForonepatientfromSenegalwe determinedthattheinfectionwascausedbyC.burnetii genotype35.InSenegal,morepatientswereinfectedwithC.burnetiiinKeurMomarSarr(p=0.002)than intheotherlocations.Bloodsamplestakenfrom500(51%males)non-febrilepeoplefromSenegaland Francewereallnegative.
Conclusions: Theinstallationofpoint-of-carelaboratoriesinruralAfricacanbeaveryeffectivetoolfor studyingtheepidemiologyofmanyinfectiousdiseases.
ß2014TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense( http://creativecommons.org/licenses/by-nc-nd/3.0/).
* Correspondingauthor.Tel.:+33491385517;fax:+33491830390. E-mailaddress:angelotasmanos@msn.com(E.Angelakis).
ContentslistsavailableatScienceDirect
International
Journal
of
Infectious
Diseases
j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / i j i d
http://dx.doi.org/10.1016/j.ijid.2014.05.029
1201-9712/ß2014TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).
consideredasignificantpublichealththreat.6 Moreover,several
strainsofC.burnetiihavebeenisolatedfromsoftticksinSenegal.6 Althoughmorethan40tickspecieshavebeenfoundinfectedwith C.burnetii,ticksarenotconsideredessentialinthenaturalcycleof C.burnetiiinlivestock.9
Tobetterguidediagnosticapproachesandempiricaltherapies inAfrica,morestudiesareneededtoassesstheroleoffastidious bacterialinfectionsinsystemicfebrileillness.Theimprovementin molecularbiologytools,especiallytheavailabilityoffullgenome sequencesandreal-time(RT)-PCRassays,hasmadeitpossibleto design specific and sensitive assays in a systematic strategic approachtothediagnosisofQfever.Theobjectiveofthisstudywas todetectthepresenceofC.burnetiiinfectioninfebrilepopulations inAfrica,anareainwhichepidemiologicalandclinicalstudiesof zoonosesarescarce,usingmolecularmethods.Wecollectedblood samplesfromfebrileandnon-febrilepatientsfromFranceandsix countries in North and Sub-SaharanAfrica toinvestigatefor C. burnetiiinfectionusingmolecularassays.
2. Materialsandmethods 2.1. Patients
PatientsfromsixAfricancountrieswithfeverwereinvestigated retrospectively for Q fever infection (Figure 1). Health centers distributedthroughoutsixruralvillagesinSenegalwereincluded fromJune 2010 to March2012. Patients weresampled in the Kenitra region (dispensaries) of Morocco in 2008,and in Mali (Diankabou,Kole)andFranceville,Gabonin2011.Patientswere sampledinSfax(infectiousdiseasesandpediatricdepartments), Tunisia,andinOran(departmentofinfectiousdiseases),Algeriain 2012.AllpatientsfromSenegal,Morocco,and Gabonoriginated fromruralareas,whereasthepatientsfromTunisia,Algeria,and Malioriginated from urbanareas (Table 1). The study sites in
Senegalcoveredvariousecosystems,fromthedryareainthenorth tothehumidarea in thesouth,witha rainyseasonfromJune through October. As controls, blood samples from non-febrile villagers in Senegal and from Marseille, France were tested. Interviews,sampling,and a medicalexamination byphysicians were performed on each individual with fever (an axillary temperature>37.58C).
2.2. Moleculardiagnosisandmulti-spacersequencetyping
A200-
m
lsampleofwholebloodwascollectedin ethylenedia-minetetraacetic acid (EDTA; Becton, Dickinson and Company, USA).Thesampleswerehandledundersterileconditionstoavoid cross-contamination.DNAwasextractedfromthebloodsamples from France, Senegal, Algeria, Tunisia, and Morocco using a QIAamp kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’sinstructions.ForGabon,DNAwasextractedfrom blood samples by DNA Blood Omega Bio-Tek E.Z.N.A method (OmegaBio-Tek, USA) asper themanufacturer’sprotocols. The genomicDNAofeachsamplewasstoredat 208Cundersterile conditions.ThegenomicDNAofeachsamplewasthensenttothe World Health Organization (WHO) Collaborative Center for Rickettsial Diseases (Marseille) under sterile conditions at 208C.DNAwasinitiallydetectedbyC.burnetii-specificRT-PCR with primersand probes designed for the amplification of the IS1111andIS30Aspacers.11ThequalityofDNAhandlingandtheextractionofsampleswasverifiedbyRT-PCRforahousekeeping gene encoding the human beta-actin gene.6,12 A sample was consideredpositive whenPCRwaspositiveforboth IS1111and IS30Aspacers.ResultswereconsiderednegativewhenPCRforC. burnetiiwasnegativeforbothIS1111andIS30Aspacersandthe cycle threshold (Ct) value of the beta-actin control gene was stronglypositive 30indicating anappropriateDNA load.DNA extractswerealsotestedbyRT-PCRforthepresenceofTropheryma
Figure1.Coxiellaburnetii-positivecasesatthedifferenthealthcentersincludedinthecurrentstudy(redcolor,ruralareas;bluecolor,urbanareas). E.Angelakisetal./InternationalJournalofInfectiousDiseases28(2014)107–110
whipplei, Bartonella spp, Rickettsia spp, Francisella tularensis, Staphylococcus aureus, Escherichia coli, and Plasmodium falci-parum.13Sampleswere alsotested by16S rRNAand 18SrRNA
geneamplificationandsequencing.13,14
We determined the multi-spacer sequence typing (MST) genotypesof C.burnetiiinthepositive C.burnetiisamplesfrom SenegalandOran,asdescribedpreviously.15
2.3. Statisticalanalysis
For data comparison, the Fisher’s exact test was performed usingEpiInfoversion6.0software(CentersforDiseaseControland Prevention,Atlanta,GA,USA).Ap-valueof0.05wasconsideredto besignificant.
2.4. Ethicsstatement
Thisstudywasdoneafterethicalapprovalwasobtainedfrom thenationalethicscommitteesofSenegal,Gabon,andFrance(No. 0-00.87MSP/DS/CNERSand No. 001380MSP/DS/CNERS).Written individualinformedconsentwasobtainedfromeachparticipant, includingtheparentsorlegalguardiansofallminors.ForMorocco, Algeria,Tunisia,andMaliourstudydesignconformedtodirectives concerningtheconductofclinicaltrialsforthesecountries. 3. Results
Overall, we tested 1238 febrile patients from Senegal (50% males),100 fromMali(50%males),50fromGabon(46%males), 184fromTunisia(59%males),268fromAlgeria(63%males),and 48fromMorocco(27%males)(Table1).Allpatientswerenegative
fortheinfectiousagentstestedexceptforC.burnetii,whichwas foundinsixfebrilepatients(0.4%)inSenegalandinone(0.3%)in Algeria(Table2).WedidnotfindpatientsinfectedwithC.burnetii inMali,Morocco,Gabon,orTunisia.FouroftheC.burnetii-positive patients(66%)fromSenegalwereadolescents(<18yearsold),one wasanadultmale,andsurprisingly,onewasa1-year-oldchild. Fever and pulmonary manifestations were the most common clinicalmanifestationsinthesepatients(Table2).Contactwaslost withthesepatientsandnonereceiveddoxycyclinetreatment.All non-febrile people from Marseille and from Dielmo and Ndiop werenegativeforC.burnetii.
In Keur Momar Sarr (Senegal),we tested 223 patients (43% males)andfoundfivefebrilepatients(2.2%;fourmalesandone female)infectedwithC.burnetii.Fourcasesoccurredduringthe dryseason,whereasonecaseoccurredduringtherainyseason.In Kedougou,wetested288patients(49%males)anddetectedone female patient (0.3%) infected with C. burnetii during the dry season.SignificantlymorepatientswereinfectedwithC.burnetiiin KeurMomarSarrthanintheotherlocationsinSenegal(p<0.001). Basedonourdata,weestimatethattheincidenceofC. burnetii-positive caseswas480/100000 febrilepatients inKeurMomar Sarr.However,wecouldnotestimatetheincidenceofC.burnetii fortheotherlocationsinSenegal.
In Oran, Algeria, we found one patient infected with C. burnetii.Thepatientwasa 21-year-oldmalewithastheniawho sufferedfromapersistentfeverandmyalgiafor6days.Thepatient had respiratory symptoms including cough, with radiographic resultscompatiblewithatypicalpneumoniaatthetimeofsample collection. Laboratory values revealed a white cell count of 9.6109/l (73% lymphocytes) and increased aspartate
amino-transferase and alanine aminotransferase (66 and 104 U/l,
Table2
PatientswithCoxiellaburnetiiinfection
Patient Place Sex Age Season Area Clinicalmanifestations Doxycyclinetreatment 1 KeurMomarSarr Male 10 Dry Rural Fever No
2 KeurMomarSarr Male 12 Dry Rural Fever No 3 KeurMomarSarr Male 15 Dry Rural Persistentfever No 4 KeurMomarSarr Male 56 Rainy Rural Persistentfeverandvomiting No 5 KeurMomarSarr Female 17 Dry Rural Feverandcough No 6 Kedougou Female 1 Dry Rural Pneumonia,vomiting No 7 Oran Male 21 Summer-dry Urban Persistentfever No Table1
Coxiellaburnetiicasesinthehealthcentersthatparticipatedinthisstudy Healthcenter Population
size No.ofpatients (%males) Climate/vegetation Positive Febrilepatients Sub-SaharanAfrica Senegal
KeurMomarSarrS3 70743 223(43%) Sahelian/steppe-type Rural 5(2.2%)
NiakharS4 69446 316(45%) Sahelo-Sudanian/woodedsteppe Rural 0
CasamanceS5 57505 411(51%) Sub-Guinean/primaryandsecondarygalleryforests Rural 0
KedougouS6 20021 288(49%) Sudano-Guinean/woodland,woodedsavannah Rural 1(0.3%)
Mali Diankabou–Kole 14833 100(50%) Sahelian/savannas,forest Urban 0 Gabon Franceville 56000 50(46%) Equatorial/savannahandtropicalforest Rural 0 NorthAfrica
Algeria Oran 1584607 268(63%) Mediterraneanclimate Urban 1(0.3%) Morocco Kenitra 1859540 48(27%) MildMediterraneanclimate Rural 0 Tunisia Sfax 2256320 184(59%) Mediterraneanclimate Urban 0
Total 1888(53%) 7
Non-febrilepatients Senegal
DielmoS1 200(53%) Sudanian/woodedsavannah 0
NdiopS2 200(40%) Sudanian/woodedsavannah 0
France Marseille 100(71%) Mediterraneanclimate 0
respectively).Thepatientreceivedtreatmentwithazithromycin for3days,andthefeverresolved.However,contactwasthenlost withthepatient.
3.1. Multi-spacersequencetyping
AlthoughsevenMSTgenotypingstudieswereattempted,only one genotype was determined; C. burnetii genotype 35 was identifiedinonepatientfromKeurMomarSarr.Allattempts to genotypetheotherpositivesampleswereunsuccessful,possibly becauseoflowDNAload.
4. Discussion
Inthisstudy,wefoundthatC.burnetiiwasthecauseoffeverin sixpatientsfromSenegaland onefromAlgeria.We believeour findingsarereliable,becauseeachpositiveresultwasconfirmedby theamplificationofasecondspecificgeneofC.burnetiitoavoid false-positiveresults.11,12AlthoughinGabonthelocallaboratory
atthetimeofthestudyusedadifferentDNAextractionassaythan theotherlaboratories,thisdidnothaveanimpactonourassays becausethequalityofDNAextractionwasverifiedforallsamples and the analysis of all controls yielded expected results. Our specificRT-PCRassayforthedetectionofC.burnetiiwascapableof detecting102bacteria/ml.11Alimitationofourstudywasthatthe
diagnosiswasbasedonlyonmolecularandnotonserologicaldata. Microimmunofluorescence (IFA) is the reference technique for diagnosingQfever.16,17Howeverthiswasnotperformedbecause IFArequireshighlyexperiencedtechnicians,9andsuchexpertise
wasnotavailableinthelocallaboratoriesthatparticipatedinthis study.Asaresult,intheabsenceofserologicaldata,wecouldnot determineifourC.burnetii-positivepatientsweresufferingfrom acuteQfeverorQfeverendocarditis.
WefoundC.burnetiigenotype35inapatientfromSenegal.This genotypewasrecentlyfoundinticksfromSenegal,6butthisisthe
firsttimethatthisgenotypehasbeendetectedinahuman.Ina previousstudy,C.burnetiigenotypes6,35,and36wereidentified in ticks from Senegal.6 Moreover, C. burnetii genotype 19 was
detectedpreviouslyinapatientwithendocarditisfromSenegal.6
Incontrast,alargerbiodiversityofstrainshasbeenobservedin samplesfrompatientsinmetropolitanFrance,where21genotypes circulate.15,18 However, in the recent Q fever outbreak in the
Netherlands,it appears that a single strain (genotype33) was responsible for the epidemic.19,20 Recently, a new C. burnetii genotype(genotype51)wasidentifiedinanaorticvalvesample fromapatientwithQfeverendocarditisinSaudiArabia.21
Inapreviousstudy,ahighseroprevalenceofQfeverwasfound inWest,Central,andsouthernAfrica.10Peopleintheseruralareas
areownersofahighnumberofdomesticruminants,andC.burnetii isalsopresentinthehouseholdenvironment.22InSenegal,ahigh
incidenceratewasrecentlyreportedamongpeoplelivinginthe villages of Dielmo (73/100 000 person-years) and Ndiop(223/ 100000 person-years).22In thepresent study,theincidenceof
acuteQfeverinKeurMomarSarrwas480/100000febrilepatients. Iftheincidenceoffeveriscomparabletothatinthevillagesof Dielmo and Ndiop, thecurrent incidence may be150/100 000 inhabitants.Ifourhypothesisistrue,Senegalmaybethecountry withthehighest Qfeverincidenceamongthetested countries. However,thefactthatwetestedfewerpatientsinMorocco,Mali, Gabon,Tunisia,andAlgeriaandthatpatientsinTunisia,Mali,and Algeriaweprimarilyfromurbanareascouldpossiblyexplainwhy wefoundonlyoneC.burnetii-positivepatientinallthesecountries. The installation of point-of-care (POC) laboratories in rural Senegalisaveryeffectivetool forstudyingtheepidemiologyof manyinfectiousdiseases.1,23 Molecularmethodsplay aprimary
role in the diagnosis of many infectious diseases, and the
widespread use of RT-PCR, which is less expensive than conventionalPCR,canreducethedelayindiagnosisofinfections. Forthisstudy,allbloodsampleswereinitiallycollectedintherural healthdispensariesandwerethensenttoourreferencecenterin Marseilleformolecularanalysis.Asaresult,alotoftimewaslostin obtainingthediagnosisandcontactwaslostwithalltheC. burnetii-infectedpatients.
Inconclusion,weshowedthatC.burnetiimaybeacauseoffever primarily in children and young adolescents living in Senegal. PatientslivinginruralareasofAfricaortravelersreturningfrom theseareaswithunexplainedfevershouldbetestedforC.burnetii infection.Molecularmethods providea convenienttool for the diagnosisofC.burnetii,andasaresult,webelievethatthepresence ofPOClaboratoriesinruralareasofAfricaiscritical.
Funding:Nofundingsourcesdeclared. Conflictofinterest:None.
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