The
Brazilian
Journal
of
INFECTIOUS
DISEASES
w w w . e l s e v i e r . c o m / l o c a t e / b j i d
Original
article
Clinical
significance
of
different
bacterial
load
of
Mycoplasma
pneumoniae
in
patients
with
Mycoplasma
pneumoniae
pneumonia
Wujun
Jiang,
Yongdong
Yan
∗,
Wei
Ji,
Yuqing
Wang,
Zhengrong
Chen
DepartmentofRespiratoryMedicine,Children’sHospitalAffiliatedtoSoochowUniversity,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3April2013
Accepted12June2013
Availableonline26October2013
Keywords:
Mycoplasmapneumoniae
Pneumonia
Polymerasechainreaction
Serology
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b
s
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c
t
Objective:Thisretrospectivestudywasconductedtoinvestigatetheclinicalsignificanceof
differentMycoplasmapneumoniaebacterialloadinpatientswithM.pneumoniaepneumonia
(MP)inchildren.
Methods:PatientswithMP(n=511)wereidentifiedattheChildren’sHospitalAffiliatedto
SoochowUniversitydatabaseduringanoutbreakofMPbetweenJanuary2012andFebruary
2013.
Results:Comparingpatientswithhighandlowbacterialloadthosewithhigherloadswere
significantlyolder(p<0.01)andhadfeversignificantlymorefrequently(p=0.01).Presence
ofwheezingatpresentationwasassociatedwithlowbacterialload(p=0.03).Baseline
posi-tiveIgMwaspresentin93(56.4%)patientswithhighbacterialloadcomparedto46(27.8%)
patientswithlowbacterialload(p<0.001).Co-infection withviruseswasfound
signifi-cantlymorefrequentamongpatientswithlowbacterialload(24.2%)thanthosewithhigh
bacterialload(8.5%)[p<0.001].Bacterialco-infectionwasalsomorefrequentlydetected
amongpatientswithlowbacterialload(22.4%)thaninthosewithhighbacterialload(12.1%)
[p=0.01].
Conclusion:M.pneumoniaeatahighbacterialloadcouldbeanetiologicagentof
respira-torytractdisease,whereastheetiologicroleofMPatalowbacterialloadremainstobe
determined.
©2013ElsevierEditoraLtda.Allrightsreserved.
Introduction
Mycoplasma pneumoniae is an important pathogen
respon-sible for community-acquired pneumonia in children.
∗ Correspondingauthorat:TheDepartmentofRespiratoryMedicine,Children’sHospitalAffiliatedtoSoochowUniversity,No.303JingDe
Road,Suzhou215003,China.
E-mailaddress:yyd3060@126.com(Y.Yan).
M.pneumoniaepneumonia(MP)hasbeenreportedin10–40%
of community-acquiredpneumoniacases.1–4 Themainstay
fordiagnosingofM.pneumoniaeinfectionpresentlyrelieson
a rise in paired titer by serological methods.5 Polymerase
chainreaction(PCR)hasbeenappliedfortheearlydetection
1413-8670/$–seefrontmatter©2013ElsevierEditoraLtda.Allrightsreserved.
of M. pneumoniae infection.6,7 The role of PCR assays
per-formedonupperrespiratorytractsamplesforthediagnosis
ofM.pneumoniaeinfectioniscontroversial.5,8 Previousstudy
demonstratedthatPCRcandetectpersistentM.pneumoniae
infection up to7monthsafterdisease onset, thebacterial
loadinconsecutivesamplesgraduallydeclinedinrelationto
thetimeintervalfromonsetofillnesstosampling.9Thus,the
clinicalsignificanceofdifferentM.pneumoniaebacterialload
isofmuchinterest.
Thisstudy wasconductedtoinvestigatetheclinical
sig-nificance of different M. pneumoniae bacterial load using
nasopharyngealaspirates(NPAs)duringarecentoutbreakof
MPinchildren.
Materials
and
methods
Patientsandspecimens
Thisstudywas aretrospectiveanalysis ofreal-time PCRfor
diagnostictestingofM.pneumoniaeinfectionbyantigen
detec-tion.Patientshospitalized withclinicallyand radiologically
confirmed lower respiratory tract infections (n=1429) were
identifiedattheChildren’sHospitalAffiliatedtoSoochow
Uni-versitydatabaseduringanoutbreakofMPbetweenJanuary
2012andFebruary2013.BothserumandNPAswereobtained
fromthesepatients.Therefore,patientswithoutstoredserum
samplesor results ofNPAs were excluded from our study.
Pairedserumsamplestakenonadmissionandatleastseven
daysafterthefirstcollectionwereavailablefor733patients
(51.3%).
Real-timePCR
Nasopharyngealswabswereobtainedwithin24hof
admis-sion. The specimens were centrifuged and were stored at
−80◦Cuntiltested.Aquantitativediagnostickit(DaAnGene
Co., Ltd., Guangzhou, China) for M. pneumoniae DNA was
usedtodeterminetheload ofMP,aspreviouslyreported.10
Themethod is basedon the TaqMan PCRtechnology, and
thetargetis16SrRNAgenespecificforMPgenome.Briefly,
1mLofnasopharyngealaspiratesdilutedwith4%NaOHwas
centrifugedat12,000rpmforfivemin.Thesedimentwas
col-lected,washedtwicewith0.9%NaCl,blendedwith50Lof
DNAextractionsolution,incubatedat100◦Cfor10min,and
centrifugedat12,000rpmforfivemin.Real-timePCRwas
per-formedontheresultingsupernatantwith2L,andPCRmix
with43L(suppliedwiththekits)usingtheDA7600real-time
PCRsystem(AppliedBiosystems,California,USA)asfollows:
93◦Cfor2min,10 cyclesof93◦Cfor45sand 55◦Cfor60s,
followedby30 cyclesof93◦Cfor30sand55◦Cfor45s.All
nasopharyngealswabswere testedforantigendetectionby
immunofluorescenceforsevencommonviruses(RSV,
adeno-virus,influenzavirusesAandB,andparainfluenzaviruses1,
2and3).
Serology
Detection of serum M. pneumoniae-specific antibody was
performed using enzyme-linked immunosorbent assay
(Virion-Serion,Germany).Theassaywasconsideredpositive
ifIgM≥1.1oriftherewas≥4-foldriseinIgGtiter.8
DiagnosisofMP
Diagnosis ofMP wasbased on serologyor PCR findings.A
significantriseinM.pneumoniaeIgGtiterorthepresenceof
IgMantibodieswasusedascriteriaforcurrentM.pneumoniae
infection.Likewise,DNAdetectionbyreal-timePCRwasalso
consideredM.pneumoniaeinfection.
Reviewofmedicalrecords
Datacompiledfromaretrospectivechartreviewofallstudy
patients were extracted through the use of specially
pre-pareddataformswithout knowledgeofserologicstatus or
PCRresults.Theinformationextractedincluded
demograph-ics,clinicalfeaturesandlaboratorydata.
Statisticalanalysis
Weusedn(%)forcategoricalvariablesandmedian(IQR)for
continuousvariableswithnon-normaldistributionormean
(SD)forthosewithnormaldistribution.Weassessed
differ-encesincategoricalvariableswiththe2test.Wecalculated
95%CIfordifferencesinmedianswithanexacttest.
Logis-tic regression analysis was performed to identify clinical
characteristicsassociatedwithdifferentbacterialloads.SPSS
(version17.0)softwarewasusedforallstatisticalanalysis.
Results
Demographicfindings
Among1429patientswithlowerrespiratorytractinfections,
511(35.8%)werediagnosedwithMPbasedonserologyorPCR
findings.Ofthe511patientswithMP,304(59.5%),themean
agewas35months(range,onemonthto156months).Theage
distributionofthepatientsisshowninFig.1.Theremaining
918patientswhowerenotdiagnosedwithMPdidnotdiffer
significantlyfrompatientswithMPintermsofsexandageat
diseaseonset. 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Ages Number of cases
200 Number of patients 150 100 50 0
Genomal bacterial loads (copies/ml) >10 7 –10 7 –10 6 –10 5 ≤10 4
Fig.2–Distributionofdifferentbacterialloadsinour patients.
QuantitativeanalysisofM.pneumoniaeDNAin
nasopharyngealaspirates
Ofthe511patientswithMP,PCRforM.pneumoniaewas
posi-tivein330(64.6%)nasopharyngealsamples.Genomalbacterial
load inNPAs rangedfrom <2500 to >2.5×107copies/mL of
sampledmaterial.Accordingtothedistributionofgenomal
bacterialloads(Fig.2),patientswereclassifiedintotwogroups:
high bacterialloadgroup (>107copies/mL)(n=165)andlow
bacterialload(<107copies/mL)(n=165).
Thegenomalbacterialloadincreasedsignificantlywithage
(p<0.001).FewerChildrenyoungerthanoneyearofagehad
lesshighbacterialload(17.7%)thanthoseolderthanfiveyears
(53.8%)[p<0.001](Fig.3).
EpidemiologyofMPoutbreak
Seasonaldistribution ofMPaccordingtogenomal bacterial
loadisshowninFig.4.
Outbreaksbeganinsummermonthsandpeaked during
AugustandSeptember(73.5%ofallMPcases).Thefrequency
ofpatientswithhighbacterialloadalsoincreasedduring
sum-merandpeakedinAugustandSeptember.FromJanuaryto
March 18 (15%)MP patients had high bacterial load,
com-paredto52(27.5%)fromApriltoJune,137(43.3%)fromJuly
toSeptember,and96(38.7%)fromOctobertoDecember.
100 80 60 40 20 0
<1y 1-2y 3-4y >5y
High bacterial load Low bacterial load PCR negative
Age of patients
Percentage
Fig.3–Differentbacterialloadsindividualscategorized accordingtoagegroup.
120
80
40
0
2012
No. of tested samples
2013
High bacterial load Low bacterial load PCR negative
1 2 3 4 5 6 7 8 9 10 11 12 1 2
Fig.4–MonthlyoccurrenceoftotalepisodesofMycoplasma pneumoniaepneumoniaandcaseswithdifferentbacterial loadsduringtheoutbreaksin2012.
ComparisonofclinicalcharacteristicsbyPCRstatus
Clinicalcharacteristics accordingtoPCRstatusare
summa-rized inTable 1. To assess the independentassociation of
evaluatedparameters,meanage,fever,andwheezingat
pre-sentationweretestedbylogisticregressionanalysis.Patients
withhighbacterialloadweresignificantlyolder(p<0.01)and
hadsignificantlyhigherfever(p=0.01).Wheezingat
presenta-tionwasassociatedwithlowbacterialload(p=0.03).
ComparisonofserologicaltestsbyPCRstatus
Out of 330 PCR positive children, 179 patients had paired
serum samples. No difference was found in the interval
betweenonsetofsymptomsandthetimeofobtainingthefirst
serum sampleor secondserumsample.93(56.4%)patients
withhighbacterialload46(27.8%)hadmoreinitialpositive
IgMcomparedtopatientswithlowbacterialload(p<0.001).
GeometricmeantitersofinitialIgGandIgMofpatientswith
highbacterialloadwerenotsignificantlydifferentfromthose
ofpatientswithlowload.Inpatientswithlowbacterialload,
geometricmean titersofsecondIgG significantlyincreased
comparedwiththeinitialIgGinpatientsmorethanfiveyears
old. In patients with high bacterial load, geometric mean
titersofsecondIgGsignificantlyincreasedinyoungerpatients
(Fig.5).Patientswithhighbacterialloadalsohadgeometric
meantitersofsecondIgMsignificantlyincreasedinyounger
patientscomparedwithpatientswithlowbacterialload.
Comparisonofco-infectionbyPCRstatus
Co-infectionsweredetectedin204patientswithMP(102with
virus and102 withbacteria).Co-infectionwithviruseswas
significantlymorefrequentamongpatientswithlow
bacte-rialloadthanthosewithhighbacterialload(24.2%vs.8.5%;
p<0.001).Bacterial co-infectionswere alsomorefrequently
detectedamongpatientswithlowbacterialloadthaninthose
withhighbacterialload(22.4%vs.12.1%;p=0.01).
Discussion
This study investigated the diagnostic values and clinical
Table1–ClinicalcharacteristicsaccordingtoPCRstatusinpatientswithMP.
Characteristics Genomalbacterialload
PCRnegative(n=181) Lowload(n=165) Highload(n=165)
Male:female(n) 109:72 100:65 95:70
Meanage(months)a 26(29) 29(31) 49(54)b,d
Durationofpriorsymptoms(days)a 7.6(2.5) 7.5(5.0) 7.2(3.7)
Antibiotictherapywithin2weeksprecedingadmission,n(%) 141(77.9) 81(84.4) 135(81.8)
Fever(Tmax≥38.5◦C),n(%) 111(61.3) 97(58.7) 127(77.0)c,d
Wheezingatpresentation,n(%) 56(30.9) 59(35.8) 26(15.8)c,d
a Dataexpressedasmeans(SD).
b p<0.01comparedwithlowgenomalbacterialload.
c p<0.05comparedwithlowgenomalbacterialload.
d Meanage,fever,andwheezingatpresentationweretestedbylogisticregressionanalysis.
M.pneumoniaeinfectionin2012.M.pneumoniaeatahighload
was more frequently detected in older children and more
stronglyassociatedwithfever,butinverselyassociatedwith
wheezing.Inaddition, positive initialIgMand significantly
increasedsecond antibodytiterswere detectedmore often
in patients with high bacterial load, which indicated that
highbacterial loadwas associatedwithincreasedhumoral
response.
VarioustestshavebeenusedtodetectMPinfection.The
mainstayfordiagnosingofM.pneumoniaeinfectionpresently
reliesonarise inIgtitersinpairedserumsamples.5
How-ever,duringtheearlyphaseofMPinfection,particularlyin
youngchildrenandimmunocompromisedpatients,
serologi-calmethodshavelowsensitivity.10,11Incontrast,PCRseemed
to be a sensitive, high-throughput and rapid method.12,13
Interestingly,thedatapresentedheredemonstratedthatthe
numberofchildrenwithhighbacterialloadincreasedwith
theage.Only17.7%ofthechildrenwithMPyoungerthanone
yearhadhighgenomalbacterialload,but53.8%ofthe
chil-drenolderthanfiveyearshadhighload.Ourstudyalsofound
thatpositiveinitialIgMweredetectedmoreofteninpatients
withhighbacterialload(56.4%vs.27.8%).Inaddition,patients
withlowbacterialloaddidneitherhaveIgMorIgGincreasein
thelessthanone-yearandonetofour-year-oldgroupwhen
serumpairedsampleswereavailable.
Fromculturebasedstudies,itisknownthatthefirststep
inthepathogenicprocessofM.pneumoniaeinvolves
cytoad-herencebetweenM.pneumoniaeandrespiratoryepithelium.
After adherence,M. pneumoniaeneeds toreplicatein order
toestablishaninfection,involvingcolonizationand
inflam-mationinhumantissues.14Theabovefindingssuggestthat
inyoungchildrenM.pneumoniaecolonizationisusuallyweak
whichleadstopoorantigenicstimulation.Thesecouldexplain
whyantibodyresponsetoM.pneumoniaeisweakordeferred
inyoungchildrenbecauseofpoorantigenicstimulationfrom
anotherprospective.
Nilssonet al.9 foundthatthe bacterialloadin
consecu-tivesamplesgraduallydeclinedinrelationtothetimeinterval
fromonsetofillnesstosampling.Infectionsassociatedwith
lowbacterialloadmayreflectM.pneumoniaepersistence.In
ourstudy,42.1%ofthePCRpositivechildrenwere
seroposi-tive.Mostoftheseseropositivediagnoses(93outof139,66.9%)
occurredinchildrenwithhighbacterialload.Highbacterial
load was notedmainly inthe absence ofother respiratory
viral or bacterial infections, suggesting acausative role of
MP.However,lowbacterialloadwasdetectedmoreoftenin
viralco-infectionandmorestronglyassociatedwith
wheez-ing,suggestingthatalowbacterialloadinthenasopharynx
seemstobeoflittleclinicalsignificance.Besides,asM.
pneu-moniaereplicatesslowlyandhaslimitedcapacityforprotein
biosynthesis, the above finding may also suggest that the
presenceofrespiratoryvirusesmayreduceM.pneumoniae
col-onization.However,wewereunabletoconfirmwhetherthis
happenedinolderchildrenduetothelowproportionofmixed
infections inagegroup(3.4% inpatientsgreater thanthree
years).
60
40
Age of patients with low bacterial loads Age of patients with high bacterial loads 20
Initial titler
Second titler Second titler
Initial titler 0 60 40 Geometr ic mean IgG Geometr ic mean IgG 20 0
<1y 1-4y >5y <1y 1-4y >5y
Fig.5–Correlationbetweeninitialandsecondantibodytitersamongpatientswithdifferentbacterialloads.Filledcircles indicategeometricmeantiters(GMTs)ofinitialantibodytitersandunfilledcirclesindicateGMTsofsecondantibodytiters ineachagegroupandbarsindicate95%confidenceintervals.
PCR tests also have their drawbacks. They could not
distinguish between viable and nonviable organisms after
antibacterial therapy.15–17 Also, concerns exist about the
potential inability to distinguish between respiratory
car-riage vs. active disease.17 Prescribing macrolides in the
presenceofapositivePCRassaymayincreasethe
unneces-saryuse ofmacrolides. Accordingtoour study,prescribing
macrolides based on a positive PCR in patients with low
bacterialloadandserologicallynegativeshouldbecarefully
considered.
Thefindingsofthepresentstudyhavesomelimitations.
Thepatients included were hospitalizedcasesof
pneumo-nia.Wedidnotenrollpatientswithotherspectrumofacute
respiratory illnesses. Therefore, patients with more severe
symptomsmayhavebeenoverrepresented.Thepatientswere
alsomorelikelytohavereceivedantibiotictreatmentdueto
theclinicalmanifestations.
Inconclusion, ourresultssuggest thatM. pneumoniaeat
ahighbacterialloadcouldbeanetiologicagentof
respira-torytractdisease,whereastheetiologicroleofMPatalow
bacterialloadremainstobedetermined.Quantitative
analy-sismay,therefore,beimportantforstudiesofM.pneumoniae
infection.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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