Chapter+2: Materials+and+Methods+
3.3 Study+cohort+
3.2.2 Recruitment+of+study+population+
A!study!lay!community!counsellor!approached!women!on!the!postnatal!ward!to!give!women!
information!about!the!study.!!Further!information!was!given!to!interested!women,!a!study!
nurse!or!Dr!C!Jones!took!informed!consent!from!eligible!women.!!Dr!C!Jones!examined!all!
infants!and!took!blood!from!each!infant.!Blood!samples!were!transported!by!Dr!C!Jones!to!
the!laboratory!at!the!University!of!Cape!town!within!4!hours!of!sample!receipt.!She!then!
prepared!the!whole!blood!culture!for!each!of!the!218!blood!samples!collected!from!mothers!
and!infants!at!birth.!The!same!procedure!was!observed!for!the!95!samples!collected!from!16!
week!old!infants.!!
!
3.3 Study+cohort+
Between!March!2009!and!January!2011,!123!eligible!motherDinfant!pairs!were!identified.!Of!
these!women,!11!declined!to!participate;!109!maternalDinfant!pairs!were!therefore!enrolled!
(91%!participation!rate).!Of!these,!47!mothers!(43%)!were!HIVDinfected!and!62!(57%)!HIVD uninfected.!All!women!testing!negative!for!HIV!at!their!antenatal!care!registration!had!a!
further!repeat!negative!HIV!test!at!delivery.!!One!infant!(1%)!was!determined!to!be!HIVD infected!at!4!weeks!of!age!and!was!referred!for!rapid!initiation!of!antiDretroviral!treatment!
(motherDinfant!pair!subsequently!excluded!from!analysis).!Of!the!original!cohort,!95!infants!
were!followed!up!to!16!weeks!of!age!(87%!of!infants).!Of!these,!39!infants!were!HIVD exposed,!uninfected!(41%)!and!56!were!HIVDunexposed!(59%).!The!number!of!samples!for!
each!assay!available!for!these!infants!is!given!in!the!relevant!results!section.!The!selection!of!
the!study!cohort!from!the!population!of!women!giving!birth!at!the!study!site!and!screened!
for!eligibility!is!shown!in!Figure!9.!!
!
! Figure+9.+Study+cohort+flow+chart+
!
The!study!cohort!was!subsequently!divided!according!to!maternal!TB!infection!status!based!
on!evidence!of!Mtb!sensitisation,!as!defined!by!a!positive!QFN!test.!The!four!groups!of!
mother/infant!pairs!were!defined!by!maternal!infection!status!namely:!HIVD!QFND!(n=27),!
HIVD!QFN+!(n=!35),!HIV+!QFND!(n=25),!HIV+!QFN!+!(n=20).!!One!HIVDinfected!mother!had!an!
indeterminate!QFN!result!(participant!data!was!used!when!considering!HIV!status!alone,!but!
not!when!considering!the!four!groups).!Cohort!clinical!data!was!analysed!only!by!maternal!
HIV!infection!status!as!this!has!most!biological!relevance.!!
!
! +
Mother+not+eligible+(n=+34)+
• <18!years!(n=!4)!
• Plans!to!leave!area!during!study!period!(n=!9)!
• Lives!outside!study!area!(n=15!)!
• Mother!unwell!(n=!3)!
• Mother!on!TB!treatment!(n=!2)!
• Current!household!contact!with!TB!(n=!1)!
Mother/infant+pairs+screened+for+eligibility+(n=+167)+
Infant+not+eligible+(n=+13)++
• Born!>24!hours!ago!(n=!1)!
• Birth!weight!<2.5kg!(n=!8)!
• Baby!unwell!(n=!1)!
• BCG!already!received!(n=3)!
!
Consent+refused+(n=+11)+
!
Eligible+mother/infant+pairs++(n=+120)+
Study+cohort+–+Enrolled+mother/infant+pairs+(n=+109)+
Completed+clinical+and+immunology+study+to+16+weeks+(n=+94)+
Loss+to+followSup+(n=+14)+
• Mother!and!infant!moved!away!(n=!6)!
• Infant!moved!away!to!live!with!relatives!(n=!4)!
• Doesn’t!wish!to!participate!further!(n=!1)!
• Unknown!(n=!3)!
+ Mother/infant+pair+excluded++
• HIVDinfected!infant!(n=1)!+
3.3.1 Maternal+Characteristics+
Characteristics!of!participating!mothers!are!given!in!Table!2.!!
!!
!! HIVDinfected!mothers!
(n=46)! HIVDuninfected!
mothers!(n=62)! p!value!
Median!age!in!years!(IQR)! 27.5!(24!–!31.25)! 24!(20D27.25)! .001!
Normal!vaginal!delivery! 46!(100%)! 62!(100%)! 1.00!
Primigravidity! 12!(26%)! 30!(48%)! .02!
BMI!at!delivery! 28.10!(25.55!D!31.69)! 27.28!(24.10!D!33.97)! .95!
BMI!at!16!weeks! 26.17!(23.47!D!28.85)! 25.60!(22.96!D!30.51)! .97!
!
Table+2.+Maternal+characteristics.++
‘Primigravidity’!refers!to!mothers!delivering!their!first!child;!BMI:!Body!Mass!Index.!!
!
The!mean!CD4!count!among!HIVDinfected!women!was!474!cells/µl!(SD!252)!and!the!median!
viral!load!730!copies/ml!(IQR!357!D!3925).!Seven!women!had!CD4!counts!<200!cells/µl;!3!of!
these!were!already!on!HAART!at!enrolment!and!a!further!4!were!referred!by!the!study!team!
to!commence!HAART!following!delivery.!!A!large!proportion!of!women!were!only!diagnosed!
with!HIV!as!a!result!of!antenatal!screening!in!this!pregnancy!(n=!27,!59%).!!
!
Most!women!received!antiretroviral!medication!as!part!of!the!PMTCT!programme;!antenatal!
AZT!or!HAART!was!received!by!89%!of!women!(n=!41),!intrapartum!NVP!or!ongoing!
treatment!with!HAART!was!administered!to!65%!of!women!(n=30).!!Six!women!were!
commenced!on!HAART!prior!to!conception!and!a!further!three!women!were!commenced!on!
HAART!in!pregnancy.!The!majority!of!infants!received!NVP!at!birth!(n!=!44,!96%)!and!
postnatal!AZT!(n=45,!98%)!for!varying!amounts!of!time!(5D28!days).!!
!
3.3.2 Infant+characteristics+
All!infants!were!fullDterm!and!of!good!weight!at!birth.!There!was!no!difference!in!growth!
between!HIVDexposed!and!unexposed!infants.!All!HIVDinfected!mothers!chose!exclusive!
replacement!feeding!and!continued!with!this!practice!throughout!the!study.!
! !
!
!
!! HIVDexposed,!uninfected!infants! HIVD!unexposed!infants! p!value!
Gestation,!weeks! 38+3!(37+1!D!39+6)! 38+4!(35+1!D!40+1)! .36!
Female! 26!(57%)! 34!(55%)! 1.00!
Birth!weight,!kg! 3.13!(2.90!D!3.45)! 3.12!(2.98!D!3.47)! .59!
Weight!at!6!weeks,!kg! 4.60!(4.20D!5.19)! 4.77!(4.49!D!5.18)! .15!
Weight!at!16!weeks,!kg! 6.39!(6.02!D!6.98)! 6.73!(6.25!D!7.40)! .16!
HC!at!birth,!cm! 34!(33!D!35.6)! 34!(33D35)! .35!
HC!at!6!weeks,!cm! 38!(37D40)! 38.3!(37.1!D!39.3)! .56!
HC!at!16!weeks,!cm! 42!(41!D!43)! 42!(41!D!43)! .98!
Length!at!6!weeks,!cm! 54!(51.9!D!56.1)! 54.3(53!D!55.9)! .51!
Length!at!16!weeks,!cm! 60.5!(58.9!D!63)! 61.5(60!D!63)! .18!
Breast!fed!at!birth! 0!(0%)! 62!(100%)! <.!001!
Breast!fed!at!6!weeks! 0!(0%)! 51!(90%)! <!.001!
Breast!fed!at!16!weeks! 0!(0%)! 37!(66%)! <.001!
!
Table+3.+Characteristics+of+participating+infants.+
‘Breast!fed’!refers!to!infants!who!received!breast!milk!either!exclusively!or!in!combination!with!
formula!milk.!Infants!below!2.5kg!at!birth!were!excluded!from!the!study.!Median!values!(IQR)!are!
stated!for!continuous!data!and!the!MannDWhitney!U!test!used!to!compare!groups.!Numbers!(%)!are!
otherwise!stated!and!groups!compared!using!Fisher’s!Exact!test.!!
!
Infants!were!screened!for!prevalent!Mtb!infection!using!a!clinical!screening!tool!and!in!
addition!IFNDγ!release!in!response!to!ESAT6/CFP10!fusion!protein!was!assessed!using!a!
standard!ELISA.!One!infant!was!admitted!to!hospital!for!a!lower!respiratory!tract!infection!at!
6!weeks!of!age!and!was!treated!for!presumed!TB.!None!of!the!other!infants!were!clinically!
suspected!of!having!TB.!Thirteeen!infants!had!a!positive!IFNDγ!response!to!ESAT6/CFP10!
fusion!protein,!however!all!that!returned!for!repeat!testing!with!a!standard!QFN!test!and!
TST!had!negative!results!and!were!clinically!well.!!
!