www.jped.com.br
ORIGINAL
ARTICLE
Reporting
detection
of
Chlamydia
trachomatis
DNA
in
tissues
of
neonatal
death
cases
夽
Maria
Hernandez-Trejo
a,b,
Norma
E.
Herrera-Gonzalez
b,
Marcos
R.
Escobedo-Guerra
c,
M.
de
Jesus
de
Haro-Cruz
c,
Elsa
R.
Moreno-Verduzco
d,
Marcela
Lopez-Hurtado
e,
Fernando
M.
Guerra-Infante
c,e,∗aDepartmentofNeurobiologyofDevelopment,InstitutoNacionaldePerinatología,DF,Mexico bSchoolofMedicine,InstitutoPolitécnicoNacional,DF,Mexico
cNationalSchoolofBiologicalSciences,InstitutoPolitécnicoNacional,DF,Mexico dDepartmentofPathologicalAnatomy,InstitutoNacionaldePerinatología,DF,Mexico eDepartmentofInfectology,InstitutoNacionaldePerinatología,DF,Mexico
Received21May2013;accepted12August2013 Availableonline30October2013
KEYWORDS
Chlamydia trachomatis; Polymerasechain reaction; Newborn; Sepsis; Mortality
Abstract
Objective: todeterminewhetherC.trachomatiswaspresentinneonateswithinfection,but
withoutanisolatedpathogen,whodiedduringthefirstweekoflife.
Methods: earlyneonataldeathcaseswhosecausesofdeathhadbeenpreviouslyadjudicatedby
theinstitutionalmortalitycommitteewererandomlyselected.End-pointandreal-time poly-merasechainreactionoftheC.trachomatisomp1genewasusedtoblindlyidentifythepresence ofchlamydialDNA intheparaffinizedsamples offiveorgans(fromauthorizedautopsies)of eachofthedeadneonates.Additionally,differentialdiagnoseswereconductedbyamplifying afragmentofthe16SrRNAofMycoplasmaspp.
Results: infivecases(35.7%),C.trachomatisDNAwasfound inoneormoreorgans.Severe
neonatalinfectionwaspresentinthreecases;oneofthemcorrespondedtogenotypeDofC.
trachomatis.Interestingly,anothercasefulfilledthesamecriteriabuthadapositivepolymerase
chainreactionforMycoplasmahominis,apathogenknowntoproducesepsisinnewborns.
Conclusion: theuseofmolecularbiologytechniquesinthesecases ofearlyinfantmortality
demonstratedthatC.trachomatiscouldplayaroleinthedevelopmentofsevereinfectionand inearlyneonataldeath,similarlytothatobservedwithMycoplasmahominis.Furtherstudyis requiredtodeterminethepathogenesisofthisperinatalinfection.
©2013SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.Allrightsreserved.
夽 Pleasecitethisarticleas:Hernandez-TrejoM,Herrera-GonzalezNE,Escobedo-GuerraMR,deHaro-CruzMJ,Moreno-VerduzcoER,
Lopez-HurtadoM,etal.ReportingdetectionofChlamydiatrachomatisDNAintissuesofneonataldeathcases.JPediatr(RioJ).2014;90:182---9.
∗Correspondingauthor.
E-mail:fguerra96@yahoo.com(F.M.Guerra-Infante).
Chlamydiatrachomatisintissuesofneonataldeathcases 183
PALAVRAS-CHAVE
Chlamydia trachomatis; Reac¸ãoemcadeiada polimerase;
Recém-nascido; Sepse;
Mortalidade
Relatodedetecc¸ãodeDNAdeChlamydiatrachomatisemtecidosdecasosdeóbito neonatal
Resumo
Objetivo: determinarseaC.trachomatisestápresenteemneonatoscominfecc¸ão,porémsem
patógenoisolado,quemorreramduranteaprimeirasemanadevida.
Métodos: casosdeóbitoneonatalprecocecujascausasdeóbito haviamsidoanteriormente
determinadaspeloComitêdeMortalidadedainstituic¸ãoforamaleatoriamente selecionados. Foramutilizadasasreac¸õesemcadeiadapolimeraseconvencionaleemtemporealdogene
omp1daC.trachomatis,paraidentificar,àscegas,apresenc¸adeDNAdeclamídianasamostras
desparafinizadasdecincoórgãos(deautópsiasautorizadas)decadaumdosneonatosmortos. Alémdisso,foramrealizadosdiagnósticosdiferenciais poramplificac¸ãodeum fragmentodo
rRNA16SdeMycoplasmassp.
Resultados: emcincocasos(35,7%)apresenc¸adeDNA deC.trachomatisfoidetectadaem
umoumaisórgãos.Haviainfecc¸ãoneonatalgraveemtrêscasos;umdelescorrespondenteao genótipoDdeC.trachomatis.Curiosamente,outrocasopreencheuosmesmoscritérios,porém possuíaumareac¸ãoemcadeiadapolimerasepositivaparaMycoplasmahominis,umpatógeno conhecidoporcausarsepseemrecém-nascidos.
Conclusão: autilizac¸ão detécnicasde biologiamolecularnoscasosde mortalidadeinfantil
precocemostrouqueaC.trachomatispoderiadesempenharumpapelnodesenvolvimentode infecc¸ãograveenoóbitoneonatalprecocesemelhanteaoobservadocomaMycoplasma homi-nis.Sãonecessáriosestudosadicionaisparadeterminarapatogênesedessainfecc¸ãoperinatal. ©2013SociedadeBrasileiradePediatria.PublicadoporElsevierEditoraLtda.Todososdireitos reservados.
Introduction
Neonatal mortality has been defined as mortality occur-ringduringthefirst28daysoflife.Itismostlyassociated to infections that are either congenital or acquired after birth.Theseinfectionsstronglyimpactthecausesofdeath and abortionin non-industrializedcountries.1,2 Chlamydia
trachomatisisthe causeofthe mostsexually transmitted
bacterial infection worldwide.3 The prevalence C.
tra-chomatis infection during pregnancy is variable: in the
United States, it is between 2% and 13.7%; in Brazil,
between 2.7% and 10%,4 and in Mexico, between 4% and
28%.5 It is related to premature rupture of membranes,
chorioamnionitis,prematurebirth,andthedevelopmentof
neonatal ophthalmitis and pneumonitis. It has also been
related to high rates of low birth weight and
perina-tal mortality.4 Variable clinical manifestations have been
observed,includingstaccatocough,prodromalrhinorrhea,
and history of conjunctivitis, tachypnea, and fever. The
most frequentfindings in chestradiographies are
intersti-tialinfiltration of bilaterallung fields,hyperinflation, and
atelectasis.6
The risk for vertical transmission of C. trachomatis is
between60%and70%,andoccursduringtheinfant’spassage
throughthe birth canal;however, thereis some evidence
thatverticaltransmissioncanalsooccurinutero,since
new-bornsdeliveredby cesarean sectionshave alsobeen born
infectedandwithintactmembranes.6---9
Recently, Rours etal.10 demonstrated the presence of
chlamydialDNAintheplacentaof pre-termproducts,and
found an association of the DNA with the degree and
progressoftissueinflammation.Currently,thereisnogood
experimentalmodelavailablefortheeffectsofChlamydia
infectionsduringpregnancyanditsassociationwith
neona-taldeath.
Aside from the pulmonary and conjunctive tissues of
newborns,Chlamydiahasalsobeenfoundinintestinal,
gen-itourinary,myocardial,andnervoussystemtissues.11---13The
presenceofChlamydiaintheseothertissuessuggeststhatit
hasaninvasivecapacity.Thepresentstudyaimedtodetect
chlamydialDNAin differenttissuesof neonateswhowere
diagnosedwith‘‘infection without an isolatedpathogen’’
and died during the first week of life. Neonates whose
evidentcauseofdeathwasnotinfectious werealso
stud-ied.Additionally, it wassought to identifythe Chlamydia
genotypesinvolvedintheseneonateswhodevelopedearly
infection.
Methods
Ethicsstatement
This study’s protocol was reviewed and approved by the institutionalEthicsCommitteeoftheSchoolMedicineatthe NationalPolytechnicInstitute,inMexicoCity,Mexico.
Definitions
Infection
Severeneonatalinfectionwasestablishedby:a)findingof
diagnosisofplacentalchorioamnionitisandpneumonitisin thecadaver.
Preterm ruptureof membranes (PROM). Rupture of the
fetalmembranespriortotheonsetoflabor, regardlessof gestationalage.14
Earlyneonataldeath. Deathoccurringbeforetheseventh
dayofextrauterinelife.15
Inclusioncriteria
The inclusion criteria were: availability of all selected organs,samples,andreports;updatedanalysisbythe peri-natalmortalitycommitteewithfinaldictum;andcomplete records.
Selectionofautopsysamples
A random selection of 20% of the cases of early neona-taldeathsthatoccurredbetweenJanuary1andDecember 31,2003andfulfilledtheinclusioncriteriawasperformed. Cases were separated into two groups according to the judgmentorfinaldiagnosisoftheinstitution’sperinatal mor-talitycommittee.Group1consistedofcasesofdeathdueto systemicinfection16withoutpathogenidentification.Group
2wasformedbycasesofdeathduetoanothercausethat
wasnotrelatedwithinfection.
The autopsy organs, specifically lungs, kidneys, brain,
liver,andbronchi,whichwereembeddedinparaffinblocks
forallparticipatingcases,werelocated.Thestudyincluded
only cases that had all these organs available, as well
as complete maternal and neonatal clinical records and
histopathologicalstudiesoftheautopsyandplacenta.The
pathologistinchiefblindlyrestudiedthehistologicsamples
fromthecadavers.
Placentas are usually routinely analyzed: hematoxylin
and eosin---stained histologic sectionsare investigated for
deciduitis, vasculitis, endometritis, or chorioamnionitis
without special studies. Later, the entire material is
dis-carded.Forthisreason,onlytheresultsofthesinglestudy
oftheplacentawereincluded.
Therestoftheplateswasdeparaffinizedandprocessed
forDNAextraction. Alltestswere performedin ablinded
manner.Clinicalanddemographicdatawereobtainedfrom
hospitalrecords.
DNAextraction
Tissue samples were deparaffinized with xylene at room temperature and washed with ethanol. The obtained tis-suewasthentreatedwithproteinaseK,andtheDNAwas obtainedby thephenol-chloroform-isoamyl techniqueand ethanolprecipitationaspreviouslydescribed.17
Chlamydiatrachomatisdetection
End-point polymerase chain reaction and real-time poly-merase chain reaction were performed using PTC-100 system (MJ Research, Inc.- Waltham, MA, United States) and StepOne (Applied Biosystems - Carlsbad, CA, United States), respectively. The primers used for end-point
polymerase chain reaction are those proposed by Dutilh
et al.18 and amplify a fragment of the omp1 gene of
C. trachomatis (5’-GCCGCTTTGAGT TCTGCTTCCTC-3’;
5’-CCAAGTGGTGCAAGGATCGCA-3’).
Eachend-pointpolymerasechainreactioncontained1.75
mMMgCl2,0.2mMdNTPs,25pMofeachproposedprimer,2.5
UTaqpolymerase(GoTaq®FlexiDNApolymerasePromega©
-Madison,WI,UnitedStates),and5LoftheDNAsample,for
afinalvolumeof25L.Thereactionmixturewasincubated
for5minat95◦C,followedby35cyclesof1minat95◦Cfor
denaturation, 1min at 59◦C for alignment, 1min at 70◦C
forextension,andafinalelongationstepof5minat70◦C.
Thesample wasconsideredpositivewhenanamplification
productof129bpwasobtained.
Thereal-timepolymerasechainreactionwasperformed
using 3 mM of 2.7 mM MgCl2, 25 pM of each of the
proposed primers, 2.5 U Taq polymerase (Applied
Biosys-tems), and 5L of the DNA sample, for a final volume
of 25L. The primers used were designed by Primer
Express 3.0 Sequence program (Applied Biosystems), the
amplified region is inside the same amplicon of 129 bp
obtainedwithDutilh’sprimersFw:5’-CCTGCTGAACCAAGC
CTTATG-3’ and Rv: 5’-AGGATCTC CGCCGAAACC-3’; probe:
5’-TCGACGGAATTC TGT-3’.The reaction mixturewas
pre-heated at95◦C for 20s, followed by40 cyclesof 30s at
95◦C,20sat60◦C,and20sat72◦C.
AmplificationanddetectionofMycoplasmaspp.for differentialdiagnoses
End-pointpolymerasechainreactionwasperformed accord-ingtotheprotocolproposedbyvanKuppeveldetal.19The
primers used were: MGSO: 5’-GCACCATCTGTCACTCTGTTA
ACCTC-3’ and GPO-1: 5’-ACTCCTACGGGAGGCAGCAGTA-3’.
Each polymerase chain reaction contained 20 pM/L
primers,10uM/LdNTPs,2mM/LMgCl2,5U/LTaq
poly-merase, 3LDNA,and 43L water, for afinal volume of
50L.Thereactionmixturewasincubatedat94◦Cfor5min,
followedby35cyclesofdenaturingat94◦Cfor1min,
align-mentat60◦Cfor1min,extensionat72◦Cfor1min,anda
finalextensionat72◦Cfor5min.Asamplewasconsidered
positiveifa715bpproductwasamplified.
To detect the species, the following primers were
used with the same polymerase chain reaction protocol:
Mychomp (5’-ATACATGCATGTCGAGCGAG-3’) and Mychomn
(5’-CATCTTTTAGTGGCGCCTTAC-3’).Additionally,M.
homi-nis was detected according to Grau et al.20 and U.
urealyticum wasdetected according toBlanchardet al.21
with the primers U5 (5’-CAATCT GCTCGTGAAGTATTAC-3’)
andU4(5’-ACGACGTCCATAAGCAACT-3’).
Analysisofrestrictionfragmentlength polymorphism
A restriction fragment length polymorphism (RFLP) anal-ysis wasperformed using a previously described method5
to genotype the samples with 129 bp amplicons. Briefly,
a 1,142 bp fragment of the C. trachomatis omp1gene
was amplified. The primers used for the amplification
were the same as those reported by Yang et al.:22
Chlamydiatrachomatisintissuesofneonataldeathcases 185
(5’-ATTTACGTGAGCAGCTCTCTCAT-3’).Samplespositivefor
the 1,142 bp amplicon were subjected to a second
polymerasechain reactionthatgeneratedan 879bp
frag-ment. The primers used were also described by Yang
et al.:22 P3 (5’-TGACTTTGTTTTCGA CCGTGTTTT-3’) and
P4(5’-TTTTCTAGATTTCATCTTGTTCAAT/CTG-3’).The879bp
fragment wasthenincubated withtheendonuclease Alu1
(Invitrogen, Applied Biosystems) for 10 h at 37◦C. The
resultingbandpatternwascomparedwiththatofthe
cor-respondingtypestrain.Inthisstudy,thatwasuW-3Cx(ATCC
VR-885D).
Results
Ofthe73casesavailable,18werechosenatrandom. How-ever,itwasnotpossibletolocatealltheorgansofinterestin threecases,andtheplacentastudyresultswerenot avail-ablefor onecase.Table 1depictsresultsofthe14 infant
mortalitycasesstudied,theperinatalcharacteristicsofthe
newborns,andthemostrelevantmaternaldata.Fourcases
fulfilledalloftheinfectionorneonatalsystemicinfection
criteria(cases 1, 3, 4,and 8). Cases 1 and 8were
nega-tiveforbacterialandfungalculturesperformedbothduring
their lifeor postmortem.On the firstday of lifefor case
3,Staphylococcushaemolyticuswasisolatedthroughblood
culture.Incase4,thenewbornlivedonlyonehour,andthe
correspondingmicrobiologicalcultureswerenotperformed.
The intentional search for DNA from Mycoplasma and
Chlamydia in postmortem tissues using polymerase chain
reactionresultedinamplificationofthe129bpC.
tracho-matisomp1geneinfiveneonates.Itwasamplifiedintwo
ormoreorgansforcases1,2,3,and4,andonlyinthe
kid-neyforcase6.Incase8,a715bpproduct,correspondingto
Mycoplasma,wasamplifiedinlungandkidneytissues(image
notshown).
Chlamydial DNA was found in tissues of cases 2 and
6, but with no clinical or histopathological correlation
indicating infection. These cases only presented with
prematurity,barotrauma,andpulmonaryhemorrhage.
Addi-tionally,amplificationof1,142bpand879bpfragmentsfor
RFLP analysisof samples positive for chlamydial DNAwas
onlyachievedforcases1,3,and4(Fig.1).TheRFLPanalysis
ofthesampleswhereamplificationofthe879bpfragment
wasachievedonlyallowedfortheidentificationofgenotype
Molecular weight marker
C– C+ P250
case 5case 4case 1case 3case 1case 3 L191 L185 K187 P185 Br187
800 pb
50 pb 1,000 pb
879 pb
Figure1 PCRorPolymerasechainreactionCRomp1geneof
Chlamydiatrachomatis879bpfragment.Deparaffinized
sam-ples of organs from autopsy. L=liver, P=lungs, K=kidneys, Br=brain,C−=Negativecontrol,C+=Positivecontrol(Numbers belongtotheinstitutionalclassificationofcases).
M Type strain Case 1
500
200 150
100
50
Figure2 Restrictionfragmentlengthpolymorphism.omp1of
ChlamydiatrachomatisgenotypeD.Sample:liverfromcase1.
Typestrain:ChlamydiatrachomatisUW-3/Cx(ATCCVR885D). Productof879bpwasrestrictedwithAluI.M,Molecularweight marker.
DofC.trachomatis(case1;Fig.2).Incases2and6,itwas
notpossibletoamplifythe1,142bpfragment,andthe
pres-enceofchlamydialDNAwasconfirmedinlivertissuethrough
real-timepolymerasechainreactiononlyforcase2(image
notshown).
All cases were endotracheally intubated and received
ventilatoryassistanceduringtheirentire life.Six casesof
prematuremembrane rupture werefound. Of these, two
neonates(cases 1and3) hadevidenceof chlamydialDNA
andremainedinuterowithrupturedmembranesfor8and
6days,respectively.Theclinicalcharacteristicsofallcases
areshowninTable2.
Thecausesofdeathintheremainingcases(cases5to7
and9to14)werenotassociatedwithinfection.Four(29%)
diedduetocausesrelatedwiththeirprematurebirth,three
(21%)due tostructuralcongenitaldefectsnot compatible
withlife,oneduetohydropsfetalisofnon-immunological
originwithseverepreeclampsia,andonedueto
maternal-fetalisoimmunization toRh. Allthese caseshad negative
polymerasechain reaction results for C. trachomatisand
Mycoplasmaspp.,withtheexceptionofcase6,previously mentioned.
Discussion
Severeinfectionsarestillthemaincauseofneonatal mor-bidityandmortalityindevelopingregions.Three-fourthsof infantmortalitycasesoccurduringthefirstweekoflife,and inmanycases,thecauseofdeathremainsunknown.23
Chlamydialinfectionintheperinatalandneonatalstage
can cause many diseases. These include conjunctivitis,
nasopharyngitis,pneumonitis,andlessfrequently,rhinitis,
middle ear otitis, myocarditis, and encephalitis.4
How-ever,thediscoveryofgeneticresiduesofthisintracellular
Hernandez-T
rejo
M
et
al.
neonataldeaths.
Case Maternalriskfactor Autopsydiagnosis Placental diagnosis
Bacterialcultures outcomes
PCR Previousdiagnosisa Newdiagnosis
1 PROMeightdays Congenitalpneumonia Chorioamnionitis Allnegative C.trachomatis
inliver,brain, andbronchus
NeonatalsepsisUEA InfectionbyC. trachomatis
2 Pretermdelivery. Previousstillbirth
Moderatepneumothorax, 40%atelectasis
Normal CSFPMPrettgeri+E gergoviae
C.trachomatis
inliverand brainand real-timePCR inliver
Pneumothorax Probableinfectionby
C.trachomatis
3 PROMsixdays Acutepneumonia,hyaline membrane
Chorioamnionitis Firstday: hemocultureS haemolyticus
C.trachomatis
inliver,kidney andbrain
NeonatalsepsisUEA InfectionbyC. trachomatis
Thirdday:CSFand hemoculturenegative 4 PROM26h
Previoustubal obstruction,abortion, andstillbirth
Severecongenital pneumonia
Necrotizing chorioamnionitis. Funisitis
NM C.trachomatis
inliverand bronchus
NeonatalsepsisUEA InfectionbyC. trachomatis
5 Previousabortion Multiplemajorstructural defects
Small Allnegative Allnegative Multiplecongenitalstructural defects
Multiplecongenital structuraldefects
6 None Renalrightagenesis
Pulmonaryhemorrhage
Normal NM C.trachomatis
inkidney
Surfactantpulmonary hemorrhage
Complicationsof prematurity 7 PROM26h 33%Hyalinemembrane
70%atelectasis
Normal Allnegative Allnegative HyalinemembranePulmonary hypertension
Complicationsof prematurity 8 PROM8h Congenitalpneumonia Severe
chorioamnionitis andfunisitis
Allnegative Mycoplasma
hominisinlung
andkidney
Congenitalneonatalsepsis UEA
Infectionby
Mycoplasmahominis
9 No Hydropsfetalis Signsofhypoxia Hemoculture:
Staphylococcusspp.
Alltheothernegative
Allnegative NonimmuneHydropsfetalis NonimmuneHydrops fetalis
10 Previousabortion Immaturitysystemic Normal NM Allnegative ImmaturityObstetrictrauma Extremeprematurity 11 Twopreviousstillbirth
andaninfantdeath
Erythroblastosis.Pulmonary emphysema
Erythroblastosis Allnegative Allnegative HydropsfetalisfromRh isoimmunization
Hydropsfetalisfrom
Rhisoimmunization 12 PROM1h Multiplemajorstructural
defects
Normal Allnegative Allnegative Multiplemajorstructural defects
Multiplemajor structuraldefects
13 None Immaturity Mildsubchorionitis
anddeciduitis
NM Allnegative Immaturity Extremeprematurity
14 Aninfantdeath Thanatophoricdysplasia Normal NM Allnegative Severebonedysplasia Severebonedysplasia
CSF,Cerebrospinalfluid;CT,Chlamydiatrachomatis;NM,notmade;PCR,polymerasechainreaction;PM,post-mortem;PROM,PretermRuptureofMembranes(18);UEA,unknownetiologic
agent.
Chlamydia
trachomatis
in
tissues
of
neonatal
death
cases
187
Table2 Clinicalcharacteristics(motherandchild)oftheinfantmortalitycases.
Case1 Case2 Case3 Case4 Case5 Case6 Case7 Case8 Case9 Case10 Case11 Case12 Case13 Case14
Age 3d7h
12m
42m 2d1h
47m
1h2m 5d20h 5m
3h1m 1d20h 48m
1d12h 46m
1d2h 45m
20m 18h 5d19m 41m 1h20m
Maternalage(years) 33 28 30 35 26 26 15 23 36 23 30 27 18 23
DurationofPROM 8d 0 6d 26h 0 0 1d2h 8h 0 0 0 1h 0 0
Delivery C C C ED C C C ED C ED C C ED DD
Gender Male Male Male Female Male Male Female Female Female Female Male Male Female Female
Apgar1and5minutes 2-5 5-4 3-7 1-2 7-8 4-7 4-9 4-8 4-8 0-1 3-7 8-9 1-1 1-1
Weight(g) 590 1,050 740 710 2,000 910 1,840 750 850 520 1,290 2,350 890 1,480
Height(cm) 29 37 33 34 44 33 43 36 31.5 30 38 45 34 32
Gestationalage(weeks) 25 29 27 26 41 26 32 25 27 25 27 32 25 31
Maternaldiagnostic Hyp PD PROMCh PROMCh IGR UTICh PROM PROM Ch
Severe Pre-eclam
PDUTI Isoimm Hydrops fetalis
PD PD
Neonatalantibiotic treatment
Ampi Amika
- Ampi
Amika
- - - Ampi
Amika
Ampi Amika
- - Dicloxa
Amika
- -
-Growthforgestational age
SGA AGA SGA AGA SGA AGA AGA AGA AGA SGA AGA LGE AGA AGA
AGA,appropriateforgestationalage;Amika,amikacin;Ampi,ampicillin;C,Cesareansection;Ch,chorioamnionitis;d,days;DD,dystocicdelivery;Dicloxa,dicloxacillin;ED,eutocic
delivery;h,hours;Hyp,hypothyroidism;IGR,intrauterinegrowthretardation;Isoimm,isoimmunization;LGE,largeforgestationalage;m,minutes;PD,pretermdelivery;Pre-eclam,
literature.24 In this study, chlamydial DNA was found in
morethan twoorgansinfourcases(cases1,2,3,and4),
whichsuggestthepotentialforamultivisceralinfectionby
thispathogen.Onenotableinstancewascase2wherethe
neonatewasbornprematurelybycesareananddieddueto
extensivepneumothorax.Case2didnothave
histopatholog-icaldataforinflammationinthelungsorplacenta;however,
the repeated finding of chlamydial DNA in the brain and
liverofthisnewbornpresentsastrongsuspicionofsystemic
infectionbyC.trachomatis.Duetothelackof
determina-tionofhistopathologicalcriteria,thiscasesuggeststhatthe
entirespectrumofC.trachomatispathogenesisisstillnot
completelyknown.
At no moment were diagnostic studies performed
regarding Chlamydia or Mycoplasma infection in these
patients,probablyduetotheirshortlifespan.Likewise,the
motherswerealsonotscreenedforatypicalpathogens
dur-ing their pregnancy. The lack of C. trachomatisinfection
studiesinthesewomenwasprobablyduetopoororno
pre-natalcare:six(43%)womenhadnoprenatalcare,five(36%)
hadonlytwoorthreeconsultations,andthree(21%)hadfive
orsixprenatalmedicalconsultations.
The causeof deathfor case 8wasasystemicinfection
byMycoplasmahominis.This pathogenhasbeenshown to
producesevereinfectioninfetusesandnewborns,andcan
beverticallytransmitted.Among theinfections causedby
Mycoplasmahominis,pneumonia,whichevolvesrapidlyto
bronchopulmonarydysplasia, andsystemic infections with
poorprognosesifnotdetectedandtreatedintime25 stand
out. This situation could also occur with the infections
causedbyC.trachomatis.
Thepolymerasechainreactionisthemostsensitiveand
rapidmethodtodetectmicrobialpathogensinclinical
spec-imens,particularly,forChlamydiaandMycoplasma,which
are difficult to culture in vitro. The application of this
methodtoclinicalspecimenshasmanypotentialpitfallsdue
tothe presence of inhibitors and contamination.Further,
the sensitivity and specificity of this assay is
depend-ent ontarget genes, primer sequences, techniques used,
DNAextraction procedures,and amplified product
detec-tion methods. However, the present investigation group
hadpreviouslystandardizedthepolymerasechainreaction
applied in this study.24 Additionally, the positive samples
intheassaywereconfirmedthroughreal-timepolymerase
chainreaction,amethodthatoffersmanygeneral
techni-caladvantages,includingreducedprobabilitiesofvariability
andcontamination,onlinemonitoring,andnorequirement
forpost-reactionanalyses.
The current capacity to detect infections such as C.
trachomatis,Mycoplasma,andUreaplasma(infectionsthat
impactthehealthofthemostvulnerablepopulations:
new-bornsandpregnantwomen),willallowfortheidentification
ofthehighfrequency bywhichthesemicroorganisms
pro-ducemembraneruptures,prematurebirths,and neonatal
diseasethatcanbecomesevereandevenfatal.6Therefore,
it is believed that, in the future, it will be necessary to
reassessthepublicpoliciesonprenatalcare.
Fiveof14casesinthisseriesreceivedempirical
antimi-crobial treatment. The antibioticscheme administered to
two of the newborns that died with systemic infections
byC. trachomatis(cases 1 and3) consisted of an
amino-glycoside and a beta-lactam, treatment that is usually
usedwhenthereissuspicionofcongenitalinfection,since
it covers almost all etiological possibilities of neonatal
infectionacquiredinutero.26However,theChlamydiaand
Mycoplasmageneraarenotincludedintheirantimicrobial
spectrum.27Ocularprophylaxiscanfailtopreventneonatal
chlamydial conjunctivitis, and does notprevent
coloniza-tionorinfectioninthelungs;theonlymeansofpreventing
chlamydialinfectioninthenewbornistreatingtheinfected
mother.
Case1didnothavebacterialor fungaldevelopmentin
theperformedcultures,andcase2wasclassifiedas
contam-inatedbecausethecerebrospinalfluidsamplewasobtained
postmortemanddevelopedthepolymicrobialspecies
Prov-idencia rettgeri and Enterobacter gergoviae, which are
not known as producers of neonatal infections. Case 3
wasdeliveredbycesarean sectionwithsixdaysof
prema-turemembranerupture andmaternal chorioamnionitis.At
birth, a sample of umbilical cordblood wasobtained for
culture, which showed the development of
Staphylococ-cushaemolyticus.This bacterium ispartof thecutaneous
floraandisnotapathogenthatproducescongenital
neona-tal infections.28 Additionally, treatment with ampicillin
andamikacindoesnotcoverStaphylococcushaemolyticus,
which is characteristically multiresistant. These premises
support theconclusion that those samples were
contami-nated.
TheserotypesofC.trachomatiswithhighinvasive
capac-itycaninvadediversetissuesofadultindividualsandcause
lymphogranuloma(L1,L2,L2a,andL3).Inthisstudy,itwas
shownthattheC.trachomatisinfectionincase1wasdueto
genotypeD,whichisoneofthehighestprevalenceserotypes
worldwideingenitourinaryinfectionsofthesexuallyactive
population.27 GenotypeDisoneofthefewC.trachomatis
serotypes withacytotoxinthathasgreathomologytothe
familyoflargecytoxins(LCTs)producedbyClostridium
dif-ficile.TheseLCTs causediversecytopathiceffectsintheir
host cells becausetheirglucosyltransferaseactivity
modi-fiestheintracellularregulatorymoleculessuchastheGTP
bindingmoleculeoftheRassuperfamily.29Additionally,LCTs
generallyinterfere withtheorganizationand dynamicsof
actininboththecytoskeletonandintracellulartrafficking.30
ThecytopathiceffectproducedbythecytotoxinofC.
tra-chomatisserotypeDresultsintheroundingofinfectedcells
duetodepolymerizationofactin.Thiscouldhelpexplain,in
part,thecapacityofserotypeDtoinfectanddisseminateto
diverseorgansinthosenewborns inwhomchlamydialDNA
wasfound.However,themechanismsthroughwhichC.
tra-chomatis infectionisdisseminated todifferentorgansare
yettobeidentified.
This study presented the possibility of infection by C.
trachomatistovariousorgansoffetusesandnewborns,and
thatthismighthavebeenassociatedwithinfantmortality.
However,furtherstudiesareneededtoconfirmthisfinding.
ThefindingofchlamydialDNAinmorethanoneorganof
autopsymaynotbethemostacceptedwayfor diagnosing
systemicinfectionorestablishingcauseofdeath.However,
in the authors’ opinion, the discovery of three different
C.trachomatisDNAsequencesbyend-point andreal-time
polymerase chain reaction and identification in one case
of the C. trachomatis genotype involved provides
suffi-cient evidence for further investigations using additional
Chlamydiatrachomatisintissuesofneonataldeathcases 189
strengthentheevidencepresentedhere,butunfortunately,
immunohistochemical stains were not performed in the
presentsamples.
Conflicts
of
interest
Theauthorsdeclaretohavenoconflictofinterests.
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