Conditions: Child Development
Keywords: infection azithromycin amoxicillin co-trimoxazole trachoma mass treatment
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 252
Healthy volunteers: yes

Brief summary:
The use of antibiotics has saved millions of human lives, however consumption of antibiotics can select for antibiotic resistant organisms and may lead to changes in commensal microbiome. This study is designed to estimate the effect of antibiotic consumption on microbiome in a rural region of rural Burkina Faso. Changes in the intestinal and nasopharyngeal microbiome and resistome following a short course of antibiotics will be measured.

Detailed description:
This study is designed to better understand the effect of a short course of antibiotics on changes in intestinal and nasopharyngeal microbiome on treated children and untreated household contacts. The investigators hypothesize that a short course of antibiotics will lead to decreased bacterial diversity shortly after completion of the antibiotic course, and higher probability of identification of bacterial resistance genes in rectal and nasopharyngeal samples. The investigators hypothesize that a 5-day course of antibiotics (azithromycin, amoxicillin, or co-trimoxazole) will lead to significantly decreased intestinal and nasopharyngeal bacterial diversity among children aged 6-59 months.

Specific Aim 1. Determine the effect of treatment with antibiotics on microbiome diversity in children aged 6-59 months following a 5-day course of antibiotics.

Specific Aim 1A. Determine the direct effect of a 5-day course of azithromycin, amoxicillin, or co-trimoxazole on intestinal and nasopharyngeal bacterial diversity in children aged 6-59 months compared to no treatment.

Specific Aim 1B. Determine the indirect effect of antibiotic treatment of children in a household on intestinal and nasopharyngeal bacterial diversity in an untreated child aged 6-59 months.

Specific Aim 1C. Assess the association between intestinal bacterial diversity and anthropometry in a population-based sample of children.

Arms:
arm 1: Comparison of nasopharyngeal and rectal microbiome in children receiving Azithromycin versus children receiving placebo Children aged 6 months to 59 months will be measured and weighed then, they will be randomized to one of the study arm and treated for 5 days.

Children will receive treatment everyday, once a day as is:

Azithromycin: 10 mg/kg once daily on Day 1, then 5 mg/kg once daily Days 2-5 arm 2: Comparison of nasopharyngeal and rectal microbiome in children receiving Amoxicillin versus children receiving placebo Children aged 6 months to 59 months will be measured and weighed then, they will be randomized to one of the study arm.

Children will receive treatment everyday, twice a day as is:

Amoxicillin: 25 mg/kg/day, divided into twice daily doses for Days 1-5 arm 3: Comparison of nasopharyngeal and rectal microbiome in children receiving Cotri-moxazole versus children receiving placebo Children aged 6 months to 59 months will be measured and weighed then, they will be randomized to one of the study arm.

Children will receive treatment everyday, once a day as is:

Co-trimoxazole: 240 mg daily for Days 1-5 arm 4: Comparison of nasopharyngeal and rectal microbiome in children receiving placebo versus children receiving antibiotics Children aged 6 months to 59 months will be measured and weighed then, they will be randomized to one of the study arm.

Children will receive Placebo everyday, once a day.

Interventions: intervention 1: Azithromycin intervention 2: Amoxicillin intervention 3: Cotrimoxazole intervention 4: Placebo
Intervention descriptions: intervention 1: Children in this arm will receive Azithromycin once a day. intervention 2: Children in this arm will receive Amoxicillin twice a day. intervention 3: Children in this arm will receive co-trimoxazole once a day. intervention 4: Children in this arm will receive Placebo once a day.

Protocol document:
Prot_000.pdf: 
A nti bi otic R esist a nc e a n d Micr o bi o m e i n C hil dr e n A g e d 1- 5 9 m o nt hs  i n N o u n a, B ur ki n a F as o 
M a n u al of O p er ati o ns a n d Pr oc e d ur es 
V ersi o n 1. 2 , M a y 1 9, 2 0 1 7 
N C T n u m ber 0 3 1 8 7 8 3 4
C e ntr e d e R ec h erc h e e n S a nt é d e N o u n a 
U ni v ersit y of  H ei d el b er g 
Fr a ncis I. Pr oct or F o u n d ati o n, U ni v ersit y of C alif or ni a, S a n Fr a ncisc o 


 
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T a bl e of C o nt e nts  
 


 
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1.  O V E R VI E W 
 
1. 1  E x ec uti v e S u m m ar y  
 
T h e us e of a nti bi otics h as s a v e d milli o ns of h u m a n li v es, h o w e v er c o ns u m pti o n of a nti bi otics 
c a n s el ect f or a nti bi otic r esist a nt or g a nis ms a n d m a y l e a d t o c h a n g es i n c o m m e ns al 
micr o bi o m e. H er e, w e pr o p os e a s eri es of st u di es d esi g n e d t o esti m at e t h e eff ect of a nti bi otic 
c o ns u m pti o n o n a nti bi otic r esist a nc e a n d micr o bi o m e i n a r e gi o n of r ur al B ur ki n a F as o. I n St u d y 
O n e, w e will m e as ur e a nti bi otic c o ns u m pti o n at t h e c o m m u nit y l e v el a n d esti m at e t h e 
r el ati o ns hi p b et w e e n c o m m u nit y-l e v el a nti bi otic c o ns u m pti o n a n d c o m m u nit y-l ev el 
p n e u m oc occ al r esist a nc e. I n St u d y T w o, w e will m e as ur e c h a n g es i n t h e i nt esti n al a n d 
n as o p h ar y n g e al micr o bi o m e a n d r esist o m e f oll o wi n g a s h ort c o urs e of a nti bi otics. 
 
1. 2  O bj ecti v es  
 
O bj ecti v e 1. D et er mi n e w h et h er c o m m u niti es wit h hi g h er l e v els of a nti bi otic c o ns u m pti o n h a v e 
hi g h er pr e v al e nc e of p n e u m oc occ al r esist a nc e i n c hil dr e n u n d er 5 y e ars of a g e. 
 
O bj ecti v e 1 B. Esti m at e t h e pr e v al e nc e of acti v e tr ac h o m a i n a r ur al r e gi o n of N ort h w est er n 
B ur ki n a F as o. 
 
O bj ecti v e 2. D et er mi n e t h e eff ect of tr e at m e nt wit h a nti bi otics o n micr o bi o m e di v ersit y i n 
c hil dr e n f oll o wi n g a 5- d a y c o urs e of a nti bi otics. 
 
O bj ecti v e 2 A.  D et er mi n e t h e dir ect  eff ect of a 5- d a y c o urs e of azit hr o m yci n, a m oxicilli n, or 
c o-tri m ox az ol e o n i nt esti n al a n d n as o p h ar y n g e al b act eri al di v ersit y i n c hil dr e n a g e d 6-5 9 
m o nt hs c o m p ar e d t o n o tr e at m e nt.  
O bj ecti v e 2 B.  D et er mi n e t h e i n dir ect eff ect of a nti bi otic tr e at m e nt of c hil dr e n i n a 
h o us e h ol d o n i nt esti n al a n d n as o p h ar y n g e al b act eri al di v ersit y i n a n u ntr e at e d c h il d a g e d 6-
5 9 m o nt hs. 
O bj ecti v e 2 C.  Ass ess t h e ass oci ati o n b et w e e n i nt esti n al b act eri al di v ersit y a n d 
a nt hr o p o m etr y i n a p o p ul ati o n- b as e d s a m pl e of c hil dr e n. 
 
1. 3  St u d y Sit es  
 
T his st u d y will t a k e pl ac e wit hi n t h e N o u n a H e alt h a n d D e m o gr a p hic S ur v eill a nc e Sit e ( H D S S). 
T h e H D S S c o v ers a n ar e a of 1, 7 7 5 k m 2, c o v eri n g 1 0 3, 5 7 6 i n di vi d u als li vi n g i n 5 8 vill a g es as w ell 
as t h e t o w n of N o u n a. F or O bj ecti v e 1 , w e will i ncl u d e 2 0 vill a g es s er v e d b y 6 h e alt h c e nt ers: 
B o ur ass o, Si k or o, L a b ar a ni, L e k u y, N o k u y B o b o, a n d K o d o u g o u i n t h e s o ut h e ast of t h e H D S S. F or 
O bj ecti v e 2 , w e will i ncl u d e t h e vill a g es of D ar a a n d K a m a d e n a i n t h e w est er n r e gi o n of t h e 
H D S S. T o a v oi d c o nt a mi n ati o n b et w e e n st u di es, t h e t w o st u d y ar e as will n ot o v erl a p. 
 
2.  B A C K G R O U N D 
 


 
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Azit hr o m yci n a n d ot h er a nti bi otics ar e b ei n g c o nsi d er e d f or r o uti n e us e i n s etti n gs wit h hi g h 
b ur d e ns of c hil d m or bi dit y a n d m ort alit y. I n B ur ki n a F as o, t h e u n d er- 5 c hil d m ort alit y r at e is 
1 1 0 p er 1, 0 0 0 li v e birt hs, w hic h is a m o n g t h e hi g h est c hil d m ort alit y r at es i n t h e w orl d. 1 T h e 
m aj or c a us es of c hil d m ort alit y i n B ur ki n a F as o ar e i nf ecti o us: u p p er r es pir at or y tr act i nf ecti o n, 
di arr h e a, a n d m al ari a. 1, 2 F urt h er m or e, m al n utriti o n is a m aj or c o ntri b ut or t o c hil d m ort alit y a n d 
m or bi dit y, wit h a n esti m at e d 3 5 % of c hil dr e n m al n o uris h e d i n r ur al B ur ki n a F as o.
3 A nti bi otics 
m a y t h er ef or e r e d uc e c hil d m or bi dit y a n d m ort alit y b y tr e ati n g u n d erl yi n g ( oft e n s u bcli nic al) 
i nf ecti o ns. W e pr o p os e t o c o n d uct t his st u dy i n a r e gi o n of r ur al B ur ki n a F as o w h er e t h e b ur d e n 
of c hil d m ort ality a n d m or bi dity is hi g h, a n d c hil d d e at hs ar e attri b ut a bl e t o u p p er r es pir at ory 
tr act i nf ecti o n, di arr h e a, m al ari a, a n d m al n utriti o n. 
 
M ass azit hr o m yci n distri b uti o n f or tr ac h o m a c o ntr ol m a y l e a d t o s u bst a nti al d ecr e as e i n c hil d 
m ort alit y. 4 A r ec e nt st u d y i n r ur al Et hi o pi a n ot e d a n e arl y 5 0 % d ecr e as e i n c hil d m ort alit y i n 
c o m m uniti es t h at h a d r ec ei v e d a si n gl e m ass azit hr o m yci n distri b uti o n f or tr ac h o m a c o ntr ol. 
T h e us e of a nti bi otics m a y als o c o ntri b ut e t o i m pr o v e d gr o wt h a n d n utriti o n o utc o m es f or 
c hil dr e n i n r e gi o ns wit h a hi g h b ur d e n of m al n utriti o n. 5 A m oxicilli n is us e d r o uti n el y as p art of 
o ut p ati e nt tr e at m e nt of ac ut e m al n utriti o n. R a n d o miz e d c o ntr oll e d tri als of a m oxicilli n as 
a dj u ncti v e t h er a p y f or m al n utriti o n h as f o u n d t h at c hil dr e n r ec ei vi n g a nti bi otics g ai n m or e 
w ei g ht o v er a n 8- w e e k p eri o d c o m p ar e d t o c hil dr e n r ec ei vi n g pl ac e b o. 6, 7 Cl ust er-r a n d o miz e d 
tri als of c o m m u nit y- b as e d m a n a g e m e nt of p n e u m o ni a wit h c o-tri m ox az ol e f o u n d si g nific a ntl y 
l o w er all-c a us e u n d er- 5 m ort alit y i n c o m m u niti es r ec ei vi n g t h e c o-tri m ox az ol e i nt er v e nti o n.8 
A m oxicilli n a n d c o-tri m ox az ol e ar e als o us e d f or pr e v e nti o n of l o w er r es pir at or y tr act i nf ecti o ns 
i n hi g h-ris k c hil dr e n.9 A r ec e nt s yst e m atic r e vi e w a n d m et a- a n al ysis of a nti bi otic us e i n c hil dr e n 
i n l o w- a n d mi d dl e-i nc o m e s etti n gs f o u n d a c o nsist e nt, str o n g ass oci ati o n b et w e e n a nti bi otic 
us e a n d r e d uc e d pr e v al e nc e of w asti n g a n d st u nti n g, b ot h a m o n g c hil dr e n wit h a n d wit h o ut 
cli nic al i n dic ati o n f or a nti bi otics. 5 W e r ec e ntl y p erf or m e d a nt hr o p o m etric a n al ys es o n c hil dr e n 
r a n d o miz e d t o azit hr o m yci n v ers us n o tr e at m e nt f or tr ac h o m a a n d f o u n d a st atistic all y 
si g nific a nt i m pr o v e m e nt i n h ei g ht a m o n g c hil dr e n tr e at e d wit h a nti bi otics ( m a n uscri pt i n 
pr e p ar ati o n).  
 
Milli o ns of d os es of azit hr o m yci n ar e distri b ut e d i n pr e d o mi n a ntl y r ur al S u b- S a h ar a n Afric a n 
s etti n gs f or tr ac h o m a c o ntr ol as p art of m ass azit hr o m yci n distri b uti o n pr o gr a ms . I n a d diti o n 
t o dr astic all y r e d uci n g t h e pr e v al e nc e of tr ac h o m a gl o b all y1 0 -1 6, pr e vi o us w or k h as s h o w n t h at 
azit hr o m yci n distri b ut e d as p art of tr ac h o m a c o ntr ol pr o gr a ms h as l e d t o d ecr e as es i n 
di arr h e a 1 7, 1 8, s ki n i nf ecti o ns, l o w er r es pir at or y i nf ecti o n1 9, a n d m al ari a2 0, i n a d diti o n t o all-c a us e 
m ort alit y. 4 O n t h e ot h er h a n d, t h e ris ks of m ass azit hr o m yci n distri b uti o n ar e l ar g el y c o nfi n e d 
t o n o n-s eri o us a d v ers e e v e nts, i ncl u di n g a b d o mi n al p ai n, n a us e a, a n d v o miti n g 2 1, a n d tr a nsi e nt 
i ncr e as e i n c o m m u nit y r esist a nc e, a r esist a nc e w hic h h as n e v er b e e n s h o w n t o b e cli nic all y 
m e a ni n gf ul a n d r esist a nc e l e v els r et ur n t o n or m al l e v els w h e n a nti bi otic s el ecti o n pr ess ur e is 
r e m o v e d.2 2, 2 3 
 
T h er e is a p ot e nti al f or u ni nt e n d e d c o ns e q u e nc es wit h t h e us e of a nti bi otics, i n cl u di n g 
r e d uc e d b act eri al di v ersit y. A nti bi otics m a y alt er t h e h ost’s c o m m e ns al micr o bi ot a. 2 2, 2 4 -2 9 A 
r ec e nt st u d y a m o n g a nti bi otic- n aï v e c hil dr e n i n Ni g er f o u n d a si g nific a nt d ecr e as e i n b act eri al 


 
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di v ersit y i n c hil dr e n r ec ei vi n g a si n gl e d os e of azit hr o m yci n c o m p ar e d t o pl ac e b o 2 4, alt h o u g h 
t his st u d y di d n ot e v al u at e l o n g-t er m c h a n g es i n micr o bi o m e f oll o wi n g a n a nti bi otic c o urs e. T o 
d at e, t h er e h a v e b e e n v er y f e w r a n d o miz e d ass ess m e nts of c h a n g es i n i nt esti n al micr o bi o m e 
f oll o wi n g a c o urs e of a nti bi otics, a n d t h us t h e c a us al c h a n g es i n micr o bi o m e ar e u n d erst u di e d. 
F urt h er m or e, a n y f u ncti o n al diff er e nc es d u e t o alt er e d micr o bi o m e r e m ai n as- y et u n k n o w n, as 
micr o bi o m e st u di es ar e i n t h eir i nf a nc y a n d m ec h a nistic st u di es ar e l ac ki n g. C hil dr e n wit h 
m al n utriti o n h a v e b e e n s h o w n t o h a v e d ecr e as e d i nt esti n al b act eri al di v ersit y. 
3 0, T his 
ass oci ati o n is t h o u g ht t o b e a r es ult of p o or di et a n d i ncr e as e d s usc e pti bilit y t o i nf ecti o n t h at 
l e a ds t o str uct ur al c h a n g es i n t h e g ut, p o or a bs or pti o n of n utri e nts, a n d s u bs e q u e ntl y alt er e d 
micr o bi o m e. 3 1 F urt h er m or e, e n vir o n m e nt al e nt eric d ysf u ncti o n is t h o u g ht t o b e wi d es pr e a d i n 
r ur al S u b- S a h ar a n Afric a. T his c o n diti o n is m e di at e d i n p art b y c hr o nic i nf ecti o n wit h gr a m 
n e g ati v e b act eri a, a n d c a us es m or p h ol o gic c h a n g es i n t h e i nt esti n es t h at l e a d t o m al a bs or pti o n 
a n d s u bs e q u e nt m al n utriti o n a n d st u nti n g. 3 2 -3 4 Pr es u m pti v e tr e at m e nt wit h a nti bi otics m a y 
m o d ul at e t h e ass oci ati o n a n d l e a d t o i nt esti n al micr o bi o m e c h a n g es t h at ar e act u all y b e n efici al 
f or c hil d h e alt h. 
 
W e pr o p os e t w o st u di es d esi g n e d t o g ai n a b ett er u n d erst a n di n g of t h e m ec h a nis ms b e hi n d 
t h e p ot e nti al b e n efits a n d c o ns e q u e nc es of a nti bi otic us e i n s etti n gs wit h hi g h c hil d m ort alit y 
a n d m or bi dit y. W e s p ecific all y pr o p os e t o st u d y t h es e o utc o m es i n c hil dr e n a g e d 6- 5 9 m o nt hs, 
as t h e y ar e b y f ar at t h e gr e at est ris k of m ort alit y d u e t o i nf ecti o us c a us es a n d ar e at t h e 
hi g h est ris k of m al n utriti o n, a n d t h us w o ul d b e n efit t h e m ost fr o m c hil d m or bi dit y a n d 
m ort alit y i nt er v e nti o ns . T o d at e, t h er e h a v e b e e n v er y f e w r a n d o miz e d ass ess m e nts of 
c h a n g es i n t h e i nt esti n al micr o bi o m e f oll o wi n g a c o urs e of a nti bi otics, a n d t h us t h e c a us al 
c h a n g es i n micr o bi o m e ar e u n d erst u di e d. W e pr o p os e t w o st u di es t o 1) u n d erst a n d t h e eff ect 
of c o m m u nit y a nti bi otic c o ns u m pti o n o n c o m m u nit y a nti bi otic r esist a nc e a n d 2) t h e eff ect of a 
s h ort c o urs e of r o uti n e a nti bi otics o n c h a n g es i n i nt esti n al a n d n as o p h ar y n g e al micr o bi o m e a n d 
r esist o m e. W e h y p ot h esiz e t h at c o m m u niti es wit h hi g h er a nti bi otic c o ns u m pti o n will h a v e 
hi g h er pr e v al e nc e of p n e u m oc occ al r esist a nc e i n n as o p h ar y n g e al s a m pl es. F urt h er m or e, w e 
h y p ot h esiz e t h at a s h ort c o urs e of a nti bi otics will l e a d t o d ecr e as e d b act eri al di v ersit y s h ortl y 
aft er c o m pl eti o n of t h e a nti bi otic c o urs e, a n d hi g h er pr o b a bilit y of i d e ntific ati o n of b act eri al 
r esist a nc e g e n es i n r ect al a n d n as o p h ar y n g e al s a m pl es. 
 
3.  S T U D Y D E SI G N  
 
W e pr o p os e t w o st u di es t o a d dr ess t h e pr o p os e d o bj ecti v es. St u d y O n e  will b e a cr oss-
s ecti o n al, o bs er v ati o n al st u d y d esi g n e d t o ass ess t h e r el ati o ns hi p b et w e e n c o m m u nit y 
a nti bi otic c o ns u m pti o n a n d p n e u m oc occ al r esist a nc e pr e v al e nc e. St u d y T w o will b e a f o ur- ar m 
r a n d o miz e d c o ntr oll e d tri al d esi g n e d t o yi el d esti m at es of t h e eff ect of a s h ort c o urs e of 
a nti bi otics o n i nt esti n al a n d n as o p h ar y n g e al micr o bi o m e di v ersit y a n d r esist o m e. 
 
3. 1  St u d y O n e: C o m m u nit y a nti bi otic c o ns u m pti o n a n d pr e v al e nc e of p n e u m oc occ al 
r esist a nc e 
 


 
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St u d y O n e is a cr oss-s ecti o n al ass ess m e nt of c o m m u nit y a nti bi otic c o ns u m pti o n a n d 
n as o p h ar y n g e al p n e u m oc occ al r esist a nc e. T h e st u d y will occ ur i n t w o p h as es.  
 
D uri n g P h as e I, w e will extr act d at a fr o m r o uti n el y-c oll ect e d Pr escri pti o n S h e et fr o m 6 h e alt h 
c e nt ers l oc at e d i n t h e N o u n a H D S S. T h es e pr escri pti o n s h e ets ar e c oll ect e d a n d st or e d at t h e 
h e alt h f acilit y f or all c hil dr e n u n d er t h e a g e of 5. T h e s h e ets ar e t a k e n t o t h e p h ar m ac y w h e n 
t h e c ar e gi v er fills a pr escri pti o n f or t h e c hil d. W e will extr act a nti bi otic pr escri pti o ns ( a n d 
dis p e ns i n g, as t h es e s h e ets ar e fill e d w h e n t h e c ar e gi v er pic ks u p t h e pr escri pti o n) f or a p eri o d 
c o v eri n g a p pr oxi m at el y 6 m o nt hs (J a n u ar y t hr o u g h J u n e, 2 0 1 7). W e will extr act t h e f oll o wi n g 
d at a o n a st a n d ar diz e d, el ectr o nic d at a c oll ecti o n f or m (s e e S ecti o n 8, D at a C oll ecti o n a n d 
M a n a g e m e nt): 
 
•  H e alt h f acilit y n a m e 
•  D at e of pr escri pti o n  
•  Vill a g e of r esi d e nc e of t h e c hil d 
•  C hil d’s a g e 
•  C hil d’s s ex  
•  A nti bi otic  
•  D os e a n d d ur ati o n of a nti bi otic 
 
T o c alc ul at e a nti bi otic c o ns u m pti o n, w e will c alc ul at e t h e t ot al d os es of a nti bi otics a n d 
st a n d ar diz e usi n g t h e siz e of t h e vill a g e p er t h e m ost r ec e nt H D S S c e ns us. 
 
D uri n g P h as e II, w e will d et er mi n e p n e u m oc occ al r esist a nc e i n a r a n d o m s a m pl e of 1 5 c hil dr e n 
a g e d 6- 5 9 m o nt hs i n e ac h vill a g e i n t h e c atc h m e nt ar e a f or e ac h h e alt h f acilit y ( e. g., e ac h vill a g e 
f or w hic h a nti bi otic d at a w er e c oll ect e d d uri n g P h as e I). C hil dr e n will b e r a n d o ml y s el ect e d f or 
i ncl usi o n b as e d o n t h e m ost r ec e nt H D S S c e ns us. N as o p h ar y n g e al s w a bs will b e c oll ect e d fr o m 
e ac h r a n d o ml y-s el ect e d c hil d, w hic h will b e pr oc ess e d vi a Kir b y- B a u er disc diff usi o n ass a y at 
t h e C R S N micr o bi ol o g y l a b or at or y (s e e S ecti o n 4 f or d et ails o n s p eci m e n c oll ecti o n a n d S ecti o n 
6 f or d et ails o n micr o bi ol o gic al m et h o ds). I n a d diti o n, w e will p erf or m c o nj u ncti v al 
p h ot o gr a p h y a n d cli nic al e v al u ati o n of t h e c o nj u ncti v a o n e ac h e nr oll e d c hil d f or r a pi d 
ass ess m e nt of tr ac h o m a pr e v al e nc e i n e ac h vill a g e, if a c hil d is di a g n os e d wit h acti v e tr ac h o m a 
t h e g u ar di a n will b e gi v e n a t u b e of t etr ac ycli n e. C ar e gi v ers of c hil dr e n r a n d o ml y s el ect e d f or 
i ncl usi o n i n t h e st u d y will a d diti o n all y c o m pl et e a bri ef a nti bi otic us e s ur v e y. 
 
3. 1. 1  S a m pl e Siz e  
 
A t ot al of 6 h e alt h f aciliti es a n d 2 0 vill a g es will b e i ncl u d e d i n St u d y O n e. W e will c oll ect 
n as o p h ar y n g e al s w a bs fr o m 3 0 0 c hil dr e n i n t h e 2 0 vill a g es. D u plic at e s w a bs will b e c oll ect e d o n 
2 c hil dr e n p er vill a g e, s o a t ot al of 3 2 0 s w a bs will b e c oll ect e d. 
 
3. 1. 2  H e alt h C e nt er a n d C o m m u nit y  Eli gi bilit y  
 


 
7  
All h e alt h c e nt ers i n t h e H D S S will b e eli gi bl e f or i ncl usi o n. W e s el ect h e alt h c e nt ers b as e d o n 
t h eir acc essi bilit y d uri n g t h e r ai n y s e as o n, a n d s el ect c o m m u niti es i n a c o nti g u o us ar e a of t h e 
H D S S. All vill a g es i n t h e c atc h m e nt ar e a of t h e h e alt h f acilit y t h at ar e i n t h e H D S S will b e 
i ncl u d e d. 
  
3. 1. 3  I n di vi d u al Eli gi bilit y  
 
C hil dr e n  r esi di n g i n t h e vill a g es i n t h e c atc h m e nt ar e a of i ncl u d e d h e alt h f aciliti es d uri n g t h e 
l ast c e ns us w h o ar e 6- 5 9 m o nt hs will b e eli gi bl e f or r a n d o m i ncl usi o n i n t h e st u d y. W e will us e 
t h e c e ns us t o r a n d o ml y s el ect 1 5 c hil dr e n a g e d 6- 5 9 m o nt hs of a g e fr o m e ac h vill a g e. If t h e 
c hil d h as m o v e d o ut of t h e vill a g e or di e d si nc e t h e l ast c e ns us, w e will r a n d o ml y s el ect a n ot h er 
c hil d fr o m t h e c e ns us. 
 
3. 2  St u d y T w o: A nti bi otic tr e at m e nt a n d micr o bi o m e c h a n g es i n c hil dr e n 
 
St u d y T w o is a r a n d o miz e d tri al of a s h ort c o urs e of a nti bi otics ( azit hr o m yci n, c o-tri m ox az ol e, or 
a m oxicilli n) c o m p ar e d t o n o tr e at m e nt. W e will ass ess t h e eff ect of a s h ort a nti bi otic c o urs e o n 
i nt esti n al a n d n as o p h ar y n g e al b act eri al di v ersit y a n d will t est f or a nti bi otic r esist a nc e i n t h e 
r esist o me. H o us e h ol ds wit h at l e ast 2 c hil dr e n a g e d 6- 5 9 m o nt hs will b e r a n d o miz e d i n a 
1: 1: 1: 1 f as hi o n t o o n e of f o ur st u d y ar ms: 1) azit hr o m yci n, 2) a m oxicilli n, 3) c o-tri m ox az ol e, or 
4) n o tr e at m e nt. All c hil dr e n mi n us o n e i n t h e h o us e h ol d will b e tr e at e d, a n d w e will ass ess t h e 
i n dir ect eff ect of a nti bi otic tr e at m e nt o n t h e u ntr e at e d c hil d r esi di n g i n t h e h o us e h ol d. W e will 
m e as ur e c h a n g es i n micr o bi o m e t hr e e d a ys f oll o wi n g c o m pl eti o n of t h e a nti bi otic c o urs e 
( pri m ar y o utc o m e). D uri n g t h e fi n al s a m pl e c oll ecti o n w e will als o c o m pl et e a bri ef s ur v e y 
a b o ut t h e c hil dr e n’s h e alt h p artic ul arl y a b o ut di arr h e a a n d r es pir at or y i nf ecti o ns. Fi n all y, 4 
w e e ks aft er t h e c o m pl eti o n of t h e tr e at m e nt w e will e v al u at e t h e c hil dr e n’s gr o wt h b y d oi n g 
a nt hr o p o m etric m e as ur e m e nts. 
 
3. 2. 1  S a m pl e Siz e  
 
W e will  e nr oll 1 2 0 h o us e h ol ds i nt o t h e f o ur st u d y ar ms ( 3 0/ ar m). 
 
3. 2. 2  B as eli n e M e as ur e m e nt 
 
Aft er e nr oll m e nt a n d i nf or m e d c o ns e nt b ut pri or t o r a n d o miz ati o n , c hil dr e n will u n d er g o 
a nt hr o p o m etr y ( h ei g ht, w ei g ht, a n d mi d- u p p er ar m circ u mf er e nc e m e as ur e m e nts). C ar e gi v ers 
will c o m pl et e a bri ef b as eli n e s ur v e y t h at will i ncl u d e i nf or m ati o n o n w h et h er t h e c hil d is 
c urr e ntl y excl usi v el y br e astf e e di n g, is c urr e ntl y ex p eri e nci n g di arr h e a ( m e as ur e d b y t h e 
st a n d ar d W H O q u esti o n, “ H as t h e c hil d h a d 3 or m or e l o os e or w at er y st o ols i n t h e pr e vi o us 2 4 
h o urs”), a n d, f or n o n- excl usi v el y br e astf e e di n g c hil dr e n, t h e c hil d’s di et ar y di v ersit y. 
 
3. 2. 3  R a n d o miz ati o n 
 


 
8  
A list of p ot e nti all y eli gi bl e h o us e h ol ds will b e g e n er at e d fr o m t h e m ost r ec e nt c e ns us. T his list 
will b e s e nt t o t h e st u d y bi ost atistici a n ( T C P), w h o will g e n er at e t h e r a n d o miz ati o n list. T h e 
r a n d o miz ati o n list will b e 1: 1: 1: 1 a n d will b e g e n er at e d wit h o ut str atific ati o n or bl oc ki n g. 
 
T h e r a n d o miz ati o n list will c o nt ai n t h e r a n d o miz ati o n ar m a n d h o us e h ol d i d e ntific ati o n n u m b er 
a n d will b e tr a ns mitt e d t o t h e fi el d c o or di n at or at C R S N.    
 
3. 2. 4  C o m m u nit y Eli gi bilit y  
 
W e will s el ect t w o c o m m u niti es t h at h a v e at l ast 1 2 0 h o us e h ol ds wit h 2 or m or e c hil dr e n u n d er 
a g e 6- 5 9 m o nt hs at t h e m ost r ec e nt c e ns us. C hil dr e n w h o ar e all er gic t o a n y of t h e st u d y 
m e dic ati o ns will n ot b e e nr oll e d i n t h e st u d y. C hil dr e n w h o ar e r ec ei vi n g a nti bi otics f or a n 
o n g oi n g dis e as e w o n’t b e i ncl u d e d i n t h e st u d y 
 
3. 2. 5  H o us e h ol d Eli gi bilit y  
 
H o us e h ol ds will b e eli gi bl e f or i ncl usi o n i n t h e st u d y if t h e y h a v e 2 or m or e c hil dr e n a g e 6- 5 9 
m o nt hs c urr e ntl y r esi di n g i n t h e h o us e h ol d.  
 
3. 2. 6  St u d y M e dic ati o n 
 
St u d y m e dic ati o n will b e pr oc ur e d l oc all y f or a m oxicilli n a n d c o-tri m ox az ol e. Azit hr o m yci n a n d 
pl ac e b o will b e pr oc ur e d i n t h e U nit e d St at es as t h e y ar e n ot r e a dil y a v ail a bl e l oc all y, a n d 
s hi p p e d t o t h e st u d y sit e. All m e dic ati o ns will b e pr e p ar e d as a n or al s us p e nsi o n.  
 
3. 2. 6. 1  D os a g e I nf or m ati o n  
 
All c hil dr e n will r ec ei v e 5 d a ys of tr e at m e nt. D os a g es will b e a d mi nist er e d o nc e p er d a y f or all 
m e dic ati o ns wit h t h e exc e pti o n of a m oxicilli n, w hic h will b e t wic e p er d a y. D os a g e will b e 
w ei g ht - b as e d, b as e d o n t h e b as eli n e w ei g ht ass ess m e nt as p art of a nt hr o p o m etr y 
m e as ur e m e nts, as f oll o ws: 
 
•  Azit hr o m yci n: 1 0 m g/ k g o nc e d ail y o n D a y 1, t h e n 5 m g/ k g o nc e d ail y D a ys 2- 5 
•  A m oxicilli n: 2 5  m g/ k g / d a y, di vi d e d i nt o t wic e d ail y d os es  f or D a ys 1- 5 
•  C o -tri m ox az ol e: 2 4 0 m g d ail y f or D a ys 1- 5 
•  Pl ac e b o: O nc e d os e d ail y f or D a ys 1- 5 
 
3. 2. 6. 2  Dir ectl y O bs er v e d Tr e at m e nt  
 
All d os es of st u d y m e dic ati o n will b e dir ectl y o bs er v e d b y a fi el d w or k er. St u d y m e dic ati o n will 
b e pr e p ar e d o n t h e d a y of a d mi nistr ati o n b y st aff at t h e C R S N a n d will b e pr e-l o a d e d i nt o 
s yri n g es wit h t h e c orr ect d osi n g b as e d o n t h e c hil d’s w ei g ht fr o m t h e b as eli n e ass ess m e nt. E ac h 
s yri n g e will b e l a b el e d wit h t h e c hil d’s i d e ntific ati o n a n d h o us e h ol d I D n u m b er. All d os es of 
m e dic ati o n will b e r ec or d e d el ectr o nic all y o n t h e t a bl et.  


 
9  
 
3. 2. 6. 3  A d v ers e E v e nts S yst e ms 
 
All st u d y m e dic ati o ns ar e g e n er all y w ell-t ol er at e d. T h e m ost c o m m o n si d e eff ects of 
azit hr o m yci n ar e di arr h e a, a b d o mi n al p ai n, a n d v o miti n g, e ac h of w hic h m a y occ ur i n f e w er 
t h a n o n e i n t w e nt y p ers o ns w h o r ec ei v e azit hr o m yci n. R ar er si d e eff ects i ncl u d e a b n or m al li v er 
f u ncti o n t ests, all er gic r e acti o ns, a n d n er v o us n ess. Di arr h e a d u e t o Cl ostri di u m difficil e  h as b e e n 
r e p ort e d i n r ar e c as es. 
E ac h d a y, t h e st aff m e m b er will scr e e n all e nr oll e d c hil dr e n f or a n y a d v ers e e v e nt. All a d v ers e 
e v e nts ( n o n-s eri o us a n d s e v er e) will b e c o m m u nic at e d t o t h e M e dic al M o nit or o n a d ail y b asis. 
 
T h e m ost c o m m o n si d e eff ects of a m oxicilli n ar e di arr h e a, a b d o mi n al p ai n, a n d v o miti n g. R ac h 
or ot h er h y p ers e nsiti vit y r e acti o ns h a v e b e e n r e p ort e d i n u p t o 1 0 % of p ati e nts. Di arr h e a d u e 
t o Cl ostri di u m difficil e  h as b e e n r e p ort e d i n r ar e c as es. 
 
T h e m ost c o m m o n si d e eff ects of c o-tri m ox az ol e (s ulf a m et h ox az ol e/tri m et h o pri m) i ncl u d e 
n a us e a, di arr h e a, a n d h e a d ac h e. S o m e p ati e nts m a y d e v el o p a r as h.  
 
D uri n g t h e c o ns e nt pr oc ess, t h e c o m m o n a d v ers e r e acti o ns t h at m a y occ ur will b e ex pl ai n e d t o 
p ar e nts/ g u ar di a ns, a n d t h e y will b e a d vis e d t o c o m m u nic at e a n y a d v ers e e v e nts t o t h e l oc al 
h e alt h f acilit y i m m e di at el y. If, f or a n y r e as o n, t h e p artici p a nt n e e ds f urt h er c ar e, t h e y will b e 
r ef err e d t o t h e n e ar est h e alt h c e nt er f or ex a mi n ati o n a n d tr e at m e nt. T h e h e alt h f acilit y w or ks 
cl os el y wit h t h e N o u n a H D S S, a n d p ati e nts w h o ar e e nr oll e d i n t h e st u d y will b e i d e ntifi e d 
b as e d o n a u ni q u e st u d y c ar d t h at will b e gi v e n t o e ac h p ar e nt/ g u ar di a n f or e ac h st u d y 
p artici p a nt. A d v ers e e v e nts will b e r e p ort e d t o t h e st u d y t e a m i m m e di at el y.    
 
3. 2. 7  St u d y Sc h e d ul e  
 
 
D a y 0  
D a y 1 - 5 
D a y 9  
4 w e e ks aft er 
tr e at m e nt 
c o m pl eti o n 
E nr oll m e nt  
X 
 
 
 
A nt hr o p o m etr y 
X 
 
 
X 
S w a bs 
X 
 
X 
 
R a n d o miz ati o n * 
X 
 
 
 
Tr e at m e nt 
 
X 
 
 
O utc o m e s ur v e y 
 
 
X 
 
* Aft er all b as eli n e ass ess m e nts ar e c o m pl et e 
 
4.  S P E CI M E N C OLL E C TI O N P R O C E D U R E S 
 
4. 1  N as o p h ar y n g e al S w a bs  
 


 
1 0  
N as o p h ar y n g e al s w a bs will b e c oll ect e d fr o m a r a n d o m s a m pl e of 1 5 c hil dr e n p er vill a g e 
i ncl u d e d i n St u d y O n e f or ass ess m e nt of a nti bi otic r esist a nc e a n d fr o m all c hil dr e n i n St u d y T w o 
f or ass ess m e nt of n as o p h ar y n g e al micr o bi o m e. 
 
T h e ex a mi n er will: 
1.  Pl ac e a p e di atric fl oc k e d s w a b wit h a n yl o n ti p t hr o u g h t h e ri g ht n ostril a n d d o w n 
t h e n as o p h ar y nx of e ac h p artici p a nt. N ot e t h at if t h e s w a b is n ot p er p e n dic ul ar t o 
t h e fr o nt al pl a n e of t h e f ac e, it is li k el y n ot i n t h e i nf eri or t ur bi n at e. 
2.  O nc e y o u r e ac h t h e n as o p h ar y nx, r ot at e t h e s w a b 1 8 0° as y o u r e m o v e t h e s w a b 
fr o m t h e n os e.  
3.  Pl ac e t h e s w a b i n a t u b e c o nt ai ni n g 1. 0 mL D N A/ R N A s hi el d m e di a b y Z y m o or S T G G 
(s ki m mil k, tr y pt o n e, gl uc os e, a n d gl yc eri n) m e di a, c ut t h e h a n dl e off usi n g st eril e 
sciss ors, a n d cl os e t h e c a p of t h e t u b e wit h t h e s w a b i m m ers e d. 
4.  T h e n as o p h ar y n g e al s w a b s a m pl es i n S T G G will i niti all y b e st or e d i n t h e fi el d at 4° C 
usi n g a n i ns ul at e d st or a g e b a g wit h Fis h er br a n d ic e g el p ac ks, a n d t h e n tr a nsf err e d 
t o - 2 0° C. T h e n as o p h ar y n g e al s w a b s a m pl es i n D N A/ R N A s hi el d m e di a will b e st or e d 
i n a m bi e nt t e m p er at ur e i n t h e fi el d. T h e n tr a nsf err e d t o a r efri g er at or or fr e ez er. 
5.  T h e sciss ors us e d t o c ut c alci u m al gi n at e s w a bs will b e st eriliz e d wit h alc o h ol p a ds or 
cl e a n e d wit h bl e ac h wi p es b et w e e n p artici p a nts. W h e n c oll ecti n g s p eci m e ns i n 
D N A/ R N A s hi el d, sciss ors will b e cl e a n e d b et w e e n p artici p a nts - first wit h bl e ac h 
wi p es, a n d t h e n wit h alc o h ol p a ds.  
 
D o n ot att e m pt t o c oll ect t h e N P s w a b if y o u ar e n ot s ucc essf ul aft er t hr e e att e m pts.  
N as o p h ar y n g e al s w a bs will b e st or e d i n D N A/ R N A s hi el d m e di a b y Z y m o or S T G G m e di a, a n d 
st a n d ar d micr o bi ol o gic t ec h ni q u es will b e us e d t o is ol at e S. p n e u m o ni a e  a n d t est f or r esist a nc e 
t o er yt hr o m yci n, t etr ac ycli n e, s ulf a-tri m et h o pri n e, oxicilli n , a n d cli n d a m yci n. R esist a nt is ol at es 
will b e ass ess e d f or t h e m ost c o m m o n g e n etic r esist a nt d et er mi n a nts ( er m B  a n d m ef A ) usi n g a 
P C R - b as e d ass a y. S er ot y p e will b e ass ess e d usi n g a n est e d P C R r e acti o n f or t h e m ost c o m m o n 
s er ot y p es, f oll o w e d b y t h e Q u ell u n g r e acti o n f or a n y u nt y p e d is ol at es.  
 
M at eri als f or S w a b C oll ecti o n f or R esist a nc e T esti n g  
 
S w a bs  
N P s p eci m e ns will b e c oll ect e d usi n g st eril e, i n di vi d u all y- wr a p p e d p e di atric fl oc k e d s w a bs wit h 
a pl astic s w a b s h aft ( m a n uf act ur e d b y C o p a n). N as al sit es will b e s w a b b e d wit h a st eril e, D acr o n 
p ol y est er-ti p p e d s w a b wit h a pl astic s h aft ( m a n uf act ur e d b y Fis h er br a n d).   
 
S a m pl e T u b es  
All fi el d s a m pl es f or D N A t esti n g will b e c oll ect e d i nt o st eril e 2. 0 ml micr oc e ntrif u g e t u b es, 
m a n uf act ur e d b y S arst e dt ®. ( D N A -fr e e t u b es will b e us e d f or c oll ecti o n i n D N A/ R N A s hi el d.) 
 
 
C o ol er B a gs wit h Fr oz e n Ic e P ac ks 


 
1 1  
I ns ul at e d c o ol er b a gs will b e us e d t o c arr y s a m pl es t o a n d fr o m t h e fi el d.  I n a d diti o n, fr oz e n g el 
ic e p ac ks d esi g n e d t o t h a w sl o wl y will b e us e d t o m ai nt ai n t h e t e m p er at ur e i n t h e c o ol er b a gs 
d uri n g tr a ns p ort. 
 
- 8 0° C Fr e ez er 
A st a n d ar d - 8 0° C fr e ez er l oc at e d at t h e C R S N micr o bi ol o g y l a b or at or y will b e us e d f or t h e 
st or a g e a n d fr e ezi n g of ic e p ac ks a n d s a m pl es. T his fr e ez er is k e pt i n a l oc k e d r o o m o n t h e 
gr o u n ds of t h e C R S N , w hic h is u n d er 2 4- h o ur s ec urit y g u ar d s u p er visi o n. 
 
Pr ot oc ol f or T u bi n g a n d H a n dli n g of S a m pl es  
 
T h e t u bi n g a n d h a n dli n g pr ot oc ol m ust b e c ar ef ull y f oll o w e d i n or d er t o pr e v e nt c o nt a mi n ati o n 
a n d e ns ur e t h e s af e tr a ns p ort of t h e s a m pl es b ac k t o t h e C R S N micr o bi ol o g y l a b or at or y a n d/ or 
t o t h e U S f or pr oc essi n g. T h e p ers o n i n c h ar g e of l a b eli n g, t u bi n g, arr a n gi n g, a n d h a n dli n g t h e 
s a m pl es n e e ds t o p erf or m t his t as k i n t h e m ost or d erl y a n d att e nti v e m a n n er. 
 
1.  B ot h h a n ds of t h e t u b er s h o ul d b e gl o v e d at all ti m es. T h e t u b er's gl o v es o nl y n e e d t o b e 
c h a n g e d w h e n a n y p ot e nti al c o nt a mi n ati o n of t h e gl o v es occ urs. T h e t u b er o p e ns t h e 
c a p p e d, hi n g e d li d of a micr oc e ntrif u g e t u b e, w hic h h as b e e n l a b el e d wit h t h e 
p artici p a nt’s r a n d o m i d e ntific ati o n n u m b er. 
2.  T h e s w a b is i ns ert e d b y t h e ex a mi n er i nt o t h e micr oc e ntrif u g e t u b e h el d b y t h e t u b er.  
T h e s w a b s h aft s h o ul d o nl y b e i ns ert e d u ntil t h e s w a b h e a d is f ull y i n t h e t u b e. T h e 
t u b er will c ut t h e s w a b s h aft wit h st eril e sciss ors.  
3.  T h e t u b er s h o ul d scr e w t h e c a p of t h e micr oc e ntrif u g e t u b e ti g htl y, flic k t h e t u b e t o mix 
t h e s a m pl e wit h t h e m e di a (f or t u b es wit h D N A/ R N A s hi el d m e di a), a n d pl ac e it i n t h e 
s a m pl e c oll ecti o n b ox, l oc at e d i n t h e c o ol er b a g fill e d wit h fr oz e n ic e p ac ks. T h e fl a p of 
t h e c o ol er b a g s h o ul d b e cl os e d b et w e e n e ac h p ati e nt.  T h e c o ol er b a g s h o ul d b e i n as 
c o ol a pl ac e as p ossi bl e i n t h e fi el d, i n a s h a d e d ar e a o ut of t h e s u n. 
 
U p o n r et ur ni n g fr o m t h e fi el d e ac h d a y, t h e s a m pl es i n S T G G will b e i m m e di at el y t a k e n t o a 
l oc al h e alt h c e nt er a n d st or e d i n a c o m m erci al - 8 0° C fr e ez er, r es er v e d s ol el y f or st or a g e of 
s p eci m e ns a n d ic e p ac ks.  All s a m pl es will b e i n s a m pl e b ox es, l a b el e d wit h t h e vill a g e n a m e f or 
e as y f ut ur e i d e ntific ati o n.  
 
4. 2  R ect al S w a bs 
 
R ect al s w a bs will b e c oll ect e d a n d pl ac e d i nt o A mi es tr a ns p ort m e di a or N or g e n St o ol 
Pr es er v ati v e. T h e t est will r e q uir e t h at t h e c hil d’s p ar e nt a n d ex a mi n ers w or k t o g et h er t o 
o bt ai n a g o o d s a m pl e.  Is it i m p ort a nt t o d escri b e t h e t est t o t h e p ar e nt s o t h at t h e y c a n b est 
assist wit h k e e pi n g t h e c hil d still d uri n g t h e pr oc e d ur e, if n ec ess ar y. 
    
I n pl ac e of st o ol s p eci m e ns, r ect al s w a bs c a n b e c oll ect e d i n t h e f oll o wi n g w a y:  
1.  P ut o n a cl e a n p air of gl o v es. 


 
1 2  
2.  P arti all y o p e n t h e f ec al s w a b p ac k a g e a n d r e m o v e t h e t o p s ecti o n of t h e c oll ecti o n vi al 
(t his c a n b e disc ar d e d). 
3.  P ositi o n t h e c hil d: 
•  Li e t h e c hil d o n his/ h er b ac k, h ol d l e gs i n t h e air (it is us ef ul t o h a v e assist a nc e). 
•  Or h a v e t h e c hil d l a y o n his/ h er st o m ac h acr oss t h e m ot h er/ g u ar di a n’s l a p 
4.  R e m o v e t h e s w a b fr o m t h e p ac k a g e. T a k e c ar e t h at t h e c ott o n ti p is n ot t o uc h e d.  If it is 
t o uc h e d, t hr o w t h e s w a b a w a y a n d b e gi n wit h a n e w o n e. 
5.  I ns ert t h e ti p of t h e s w a b i nt o t h e c hil d’s a n us o nl y as f ar as n e e d e d t o c o nt act f ec al 
m at eri al ( 1- 3c m) a n d r ot at e 1 8 0 d e gr e es.  T h e ti p s h o ul d b e a br o w nis h c ol or w h e n 
r e m o v e d. 
6.  Pl ac e s w a b i nt o t h e pr es er v ati v e i n t h e c oll ecti o n t u b e. M a k e s ur e t h e s w a b is f ull y 
s u b m er g e d i n t h e li q ui d pr es er v ati v e a n d t h e n br e a k t h e s w a b off usi n g t h e pr e-sc or e d 
br e a ki n g p oi nt.  
7.  Scr e w t h e c a p b ac k o n t h e t u b e a n d m a k e s ur e t h at it’s ti g ht e n e d. Wr a p t h e ar e a w h er e 
t h e c a p m e ets t h e t u b e wit h P ar afil m t o e ns ur e t h at t h e s a m pl e will n ot l e a k, a n d t h e n 
pl ac e t h e t u b e i nt o t h e a p pr o pri at e s a m pl e b ox.  
•  If t h e s w a b c a n n ot b e br o k e n off w hil e t h e ti p is f ull y s u b m er g e d i n t h e li q ui d, tr y 
t wirli n g t h e s w a b i n t h e li q ui d first (t o r el e as e t h e c o nt e nts of t h e s a m pl e i nt o 
t h e pr es er v ati v e) b ef or e br e a ki n g it off. A v oi d r u b bi n g t h e s a m pl e o n t h e ti p of 
t h e s w a b off o n t h e si d e of t h e t u b e w h er e t h er e is n o li q ui d. 
8.  Pl ac e a r a n d o m n u m b er l a b el o n t h e c oll ecti o n t u b e. 
9.  Pl ac e t h e t u b e t h e r ect al s w a b c o nt ai n er. 
1 0.  S w a b st or a g e f or G e n etic a n al ysis:  St or e s a m pl es at r o o m t e m p er at ur e. Acc or di n g t o 
t h e m a n uf act ur er, t h e pr es er v ati v e i n t h e t u b e will pr es er v e D N A f or 5 m o nt hs at r o o m 
t e m p er at ur e ( 7 d a ys f or R N A), a n d t h er e aft er c a n b e fr oz e n (-2 0° C or -8 0° C) f or l o n g -
t er m st or a g e. 
 
R ect al S w a b C oll ecti o n f or C ult uri n g/ Micr o bi ol o gic al T esti n g  
 
S w a b  
A n i n di vi d u all y- wr a p p e d C o p a n fl oc k e d s w a b wit h a pl astic s h aft will b e us e d t o c oll ect t h e 
r ect al s w a b a n d t h e n pl ac e d i nt o a St o ol N ucl eic Aci d C oll ecti o n a n d Tr a ns p ort T u b e c o nt ai ni n g 
N or g e n St o ol Pr es er v ati v e or A mi es Tr a ns p ort M e di u m.   
 
S a m pl e T u b e wit h M e di a 
T h e s p eci m e n will b e i n a st eril e St o ol N ucl eic Aci d C oll ecti o n a n d Tr a ns p ort T u b e c o nt ai ni n g 
N or g e n St o ol Pr es er v ati v e or A mi es Tr a ns p ort M e di u m wit h a c a p t h at will b e ti g ht e n e d fir ml y.  
 
Q u alit y C o ntr ol M e as ur es f or S p eci m e n C oll ecti o n 
 
N e g ati v e Fi el d C o ntr ols  
N e g ati v e fi el d c o ntr ol s w a bs f or N P a n d st o ol will b e t a k e n i n e ac h c o m m u nit y t o ass ess f or 
c o nt a mi n ati o n: o n e co ntr ol s w a b e ac h ( N P a n d st o ol/r ect al) ar e t a k e n b ef or e s p eci m e n 
c oll ecti o n b e gi ns i n a c o m m u nit y; a n d a n ot h er ( N P a n d st o ol/r ect al) u p o n c o m pl eti o n of 


 
1 3  
s p eci m e n c oll ecti o n. F or e ac h n e g ati v e fi el d c o ntr ol, t h e ex a mi n er will o p e n a n e w s w a b as 
d escri b e d a b o v e. W a v e t h e s w a b i n t h e air, wit h o ut m a ki n g c o nt act wit h a n y o n e/ a n yt hi n g.  
T u b e t h e s w a b i n m e di a, as d escri b e d a b o v e.  
 
D u plic at e s w a bs  
D u plic at e N P s w a bs a n d r ect al s w a bs/st o ol s p eci m e n will b e c oll ect e d fr o m t w o c hil dr e n p er 
c o m m u nit y.  
 
St o ol s a m pl es  
 
E ac h c ar e gi v er will b e s u p pli e d wit h a b a g a n d pl astic c hil d’s p ott y c h air a n d i nstr uct e d t o h a v e 
t h eir c hil d d ef ec at e i n a b a g a n d r et ur n t h e b a g t o t h e st o ol c oll ecti o n st ati o n. C hil dr e n t h at 
c a n n ot pr o d uc e a st o ol wit hi n t w o h o urs will b e gi v e n s u p pli es t o c oll ect t h e st o ol at h o m e. 
C ar e gi v ers will b e i nstr uct e d t o c oll ect st o ol t h e f oll o wi n g m or ni n g, aft er w hic h a st u d y t e a m 
m e m b er w o ul d c o m e t o t h eir h o us e t o pic k u p t h e s p eci m e n. Fr o m a si n gl e st o ol s p eci m e n, w e 
will pr es er v e t w o s a m pl es: 0. 5 ml of st o ol i n 0. 5 ml et h a n ol a n d 1 gr a m of st o ol i n 1 0 ml s o di u m 
ac et at e- ac etic aci d f or m ali n ( S A F) f or micr osc o p y. St o ol s a m pl es pr es er v e d i n et h a n ol a n d 
p ot assi u m dic hr o m at e s a m pl es will b e pl ac e d o n ic e i m m e di at el y aft er c oll ecti o n a n d 
tr a ns p ort e d t o a - 2 0º C fr e ez er b y t h e e n d of t h e d a y. S A F- pr es er v e d s a m pl es will b e k e pt at 
r o o m t e m p er at ur e i n t h e s h a d e. 
 
S u p pli es f or st o ol s a m pl e c oll ecti o n 
 
•  C hil d p ott y  
•  W o o d e n d o wls  
•  Di git al s p eci m e n sc al e 
•  St o ol s p eci m e n t u b e c o nt ai ni n g 1 0 ml S A F pr es er v ati v e 
•  2 ml t u b es f or P C R pr es er v ati o n 
•  A p plic at or stic k 
•  Alc o h ol wi p e  
•  Fi n e p er m a n e nt M ar k er 
•  Tr as h b a g 
•  Gl o v es  
•  B att eri es 
•  S a nitizi n g h a n d wi p es  
 
L a b or at ory m et h o ds 
 
a. R e al-ti m e t ar g et e d P C R  
D N A  will  b e  extr act e d  fr o m  r ect al  s w a bs  a n d  st o ol  s a m pl es  usi n g  t h e  t h e  Z R- 9 6  D N A  Cl e a n  & 
C o nc e ntr at or ™- 5  Kit  ( Z y m o  R es e arc h  C or p or ati o n,  Ir vi n e,  C A).  D N A  c o nc e ntr ati o n  will  b e 
m e as ur e d  usi n g  t h e  Q u bit  1. 0  Fl u or o m et er  (Lif e  T ec h n ol o gi es,  C arls b a d,  C A)  a n d  t h e  Q u bit 


 
1 4  
ds D N A  Br o a d  R a n g e  Ass a y  Kit  (Lif e  T ec h n ol o gi es).  Q u a ntit ati v e  r e al-ti m e  p ol y m er as e  c h ai n 
r e acti o n ( q P C R) will b e p erf or m e d t o f or s p eci ati o n a n d q u a ntific ati o n of t h e p ar asit e b ur d e n as 
pr e vi o usl y d escri b e d b y Pil o ett e et al 1 8. S p eci es-s p ecific q P C R a m plific ati o n will b e c o n d uct e d i n 
7 μl v ol u m es, c o nt ai ni n g 3. 5 μl of 2 X T a q M a n F ast U ni v ers al P C R M ast er Mix (Lif e T ec h n ol o gi es), 
1 2 5 n m ol of e ac h ass a y’s r es p ecti v e pr o b e, 2 μl of t e m pl at e D N A at a c o nc e ntr ati o n of 1 n g/ μl, 
a n d r es p ecti v e c o nc e ntr ati o ns of e ac h s p eci es-s p ecific P C R r e acti o n d e v el o p e d b y Pil ott e et al 
(s e e  T a bl e  2). T h er m oc ycli n g  c o n diti o ns  will  b e  as  f oll o ws:  a n  i niti al  2  mi n  i nc u b ati o n  st e p  at 
5 0° C, f oll o w e d b y a 1 0 mi n i nc u b ati o n at 9 5° C. T h es e i nc u b ati o ns will b e f oll o w e d b y 4 0 c ycl es 
of 9 5° C f or 1 5 s ec f or d e n at ur ati o n, f oll o w e d b y 1 mi n at 5 9° C f or a n n e ali n g a n d ext e nsi o n. All 
r e acti o ns will b e c o n d uct e d usi n g t h e St e p O n e Pl us R e al- Ti m e P C R S yst e m (Lif e T ec h n ol o gi es). 
 
b. Micr osc o py  
All  s o di u m  ac et at e- ac etic  aci d  f or m ali n  pr es er v e d  s a m pl es  will  b e  pr oc ess e d  at  t h e  C R S N 
l a b or at or y  usi n g  t h e  di et h yl  et h er-c o nc e ntr ati o n  m et h o d  a n d  ex a mi n e d  micr osc o p y  f or  s oil-
tr a ns mitt e d  h el mi nt h  e g gs  a n d  l ar v a2 4.  Bri efl y, t h e  pr es er v e d  s p eci m e n  will  b e  g e ntl y  s h a k e n, 
t h e n  filt er e d  a n d  c e ntrif u g e d  f or  o n e  mi n ut e  at  2 0 0 0  r e v ol uti o ns  p er  mi n ut e  (r p m).  T h e 
s u p er n at a nt  will  b e  d ec a nt e d  a n d  t h e  pr eci pit at e  mix e d  wit h  7  ml  of  0. 8 5 %  N a Cl  a n d  3  ml  of 
di et h yl et h er. T h e w h ol e s a m pl e will b e c e ntrif u g e d a g ai n f or fi v e mi n ut es at 2 0 0 0 r p m 2 4, t h e 
s u p er n at a nt  d ec a nt e d  a g ai n,  a n d  t h e  s e di m e nt  ex a mi n e d  wit h  a  micr osc o p e  at  1 0x  a n d  4 0x 
m a g nific ati o n  f or  h el mi nt h  e g gs  a n d  i nt esti n al  pr ot oz o a  c ysts.  F or  h el mi nt hs,  t h e  n u m b er  of 
e g gs  will  b e  c o u nt e d  a n d  r ec or d e d,  wit h  a  p ossi bl e  r a n g e  of  1  u p  t o  > 1 0 0  e g gs.  As  q u alit y 
c o ntr ol, a n i n d e p e n d e nt ex p ert l a b or at or y t ec h nici a n will c o nfir m all p ositi v e sli d es, as w ell as 
e v er y 1 0 t h n e g ati v e s p eci m e n.  
 
4. 3  C o nj u ncti v al P h ot o gr a p h y  a n d Cli nic al e v al u ati o n.  
 
4. 3. 1 E x a mi n ati o n p ositi o n  
T h e ex a mi ni n g p ositi o n t o b e us e d i n t h e fi el d f or y o u n g c hil dr e n will b e t h e cl assic p e di atric 
o p ht h al mic ex a mi n ati o n t ec h ni q u e. Wit h t h e ai d of a h el p er s e at e d dir ectl y o p p osit e t o t h e 
ex a mi n er, t h e c hil d will b e p ositi o n e d wit h his/ h er h e a d b et w e e n t h e ex a mi n er's k n e es, wit h 
t h e c hil d's f ac e l o o ki n g u p w ar ds t o w ar d t h e ex a mi n er.  T h e l e gs of t h e c hil d will b e str a d dl e d 
acr oss t h e h el p er a n d t h e ar ms h el d g e ntl y acr oss t h e c hil d’s c h est.  C ar e s h o ul d b e t a k e n t o 
k e e p t h e c hil d’s e y es a b o v e t h e l e v el of t h e ex a mi n er’s k n e es, i n or d er t o pr o p erl y t a k e t h e 
c o nj u ncti v al s w a b. 
 
4. 3. 1 E v erti n g t h e u p p er e y eli d  
F or all p artici p a nts, o nl y t h e ri g ht u p p er e y eli d will b e ex a mi n e d f or t his st u d y.  T h e o nl y 
exc e pti o n t o t his is if t h e ri g ht e y e is diffic ult t o ex a mi n e d u e t o e y e dis e as e or i nj ur y, i n w hic h 
c as e t h e l eft e y e will b e ex a mi n e d. I n or d er t o a v oi d p assi n g c o nt a mi n ati o n fr o m t h e c hil d i nt o 
t h e e y e, o nc e t h e ex a mi n er d o ns a n e w p air of gl o v es, t h eir gl o v e d h a n ds s h o ul d n ot b e us e d t o 
p osit i o n t h e c hil d. T h e ex a mi n er us es t h eir fi n g erti ps t o gr as p t h e c e ntr al p orti o n of t h e 
p artici p a nt’s u p p er li d e y el as h es. T h e u p p er li d is t h e n e v ert e d, usi n g a fi n g er of t h e ex a mi n er's 
ot h er h a n d ( or t h e e n d of a st eril e s w a b) as a f ulcr u m, p ositi o n e d s u p e rior t o t h e t ars al pl at e. 


 
1 5  
T h e e v ert e d li d is h el d i n pl ac e b y t h e ex a mi n er's n o n- d o mi n a nt h a n d h ol di n g t h e e y el as h es 
a g ai nst t h e or bit al ri m. 
 
4. 3. 3 C o nj u ncti v al p h ot o gr a p h y  
S p ecific  t e a m  m e m b ers  will  b e  d esi g n at e d  as  p h ot o gr a p h ers  f or  t h e  e ntir e  tri al  i n  or d er  t o 
e ns ur e hi g h q u alit y p h ot o gr a p hs b y a n ex p eri e nc e d p h ot o gr a p h er. T h e p h ot o gr a p h er will t a k e 
p h ot o gr a p hs  of  t h e  c o nj u ncti v a  wit h  a  h a n d h el d  Ni k o n  D 9 0  di git al  c a m er a  wit h  a  m acr o  l e ns 
( 1: 1). C o nj u ncti v al p h ot o gr a p h y c a us es n o d a m a g e t o t h e e y e, is w ell t ol er at e d b y c hil dr e n, a n d 
is  a  st a n d ar d  cli nic al  pr oc e d ur e  at  U C S F.  P h ot o gr a p hs  will  b e  i d e ntifi e d  b y  first  t a ki n g  a 
p h ot o gr a p h of t h e 6- di git r a n d o m i d e ntific ati o n stic k er t h at w as assi g n e d t o t h e c hil d d uri n g t h e 
r e gistr ati o n p h as e of t h e st u d y visit. B y c o n v e nti o n, all p h ot os t a k e n aft er t his r a n d o m n u m b er 
( u p t o t h e n ext p h ot o gr a p h e d r a n d o m n u m b er) b el o n g t o t h e pr ec e di n g r a n d o m n u m b er. If a 
r a n d o m n u m b er is mist a k e nl y n ot p h ot o gr a p h e d b ef or e t a ki n g a p h ot o gr a p h of t h e c o nj u ncti v a, 
it m a y b e p h ot o gr a p h e d aft er w ar ds; i n t his c as e, a n ot e s h o ul d b e i ncl u d e d t h at s a ys “ pr ec e di n g 
p h ot os.” P h ot o gr a p hs ar e st or e d o n t h e m e m or y c ar d i n t h e c a m er a, a n d t h e n tr a nsf err e d t o a n 
ext er n al h ar d dri v e e ac h ni g ht. B att eri es ar e c h ar g e d e ac h ni g ht; e ac h c a m er a b a g h as at l e ast 2 
s p ar e b att eri es. 
 
P h ot o gr a p h y Pr ot oc ol  
§  E q ui p m e nt n e e d e d: c a m er a, l a b els, p h ot o l o g b o o k, a n d C D b ur n er 
§  Misc ell a n e o us e q ui p m e nt: Extr a b att er y, c ar d r e a d er, l e ns filt er, extr a m e di a c ar d  
 
C a m er a S et u p  
A  h a n d h el d  Ni k o n  D-s eri es  c a m er a  a n d  l e ns  will  b e  us e d  f or  all  p h ot o gr a p hs.  If  p ossi bl e,  us e 
n at ur al li g ht r at h er t h a n a fl as h, as t h e fl as h i ntr o d uc es artif acts i nt o t h e i m a g e.  
§  M o d el 
o  Ni k o n D -s eri es c a m er a: D-4 0, 5 0, 7 0, 8 0, 9 0, 1 0 0 or 2 0 0  
o  T h er e  ar e  mi n or  diff er e nc es  b et w e e n  t h e  diff er e nt  m o d els,  m ostl y  r el at e d  t o 
s et u p 
§  L e ns 
o  Ni k o n M acr o A ut of oc us 1 0 5 m m f/ 2. 8 
§  S etti n gs 
o  M a n u al s etti n g- t ur n off a ut of oc us 
o  Dis e n g a g e “li mit”  e n g a g e “f ull” 
o  Ext e n d l e ns t o 1: 1; n ot e wit h ol d er v ersi o ns of t his l e ns it is 1: 1 at f ull ext e nsi o n; 
n e w er v ers i o ns c a n g o t o sli g htl y hi g h er m a g nific ati o n t h a n 1: 1 s o c a uti o n m ust 
b e ex ert e d 
o  I m a g e siz e/r es ol uti o n 
§  J p g n or m al- s et t hr u “s et u p” m e n u 
§  L ar g e-  s et  t hr u  “s et u p”  m e n u  (“s m all  a n d  m e di u m”  ar e  pr o b a bl y 
a d e q u at e) 
§  W hit e b al a nc e- a ut o m atic or fl as h; s et t hr u “s et u p” m e n u 
§  S h utt er- A p ert ur e Pri orit y 
§  f 5 7- s et t hr u m e n u; n ot e if l e ns is n ot ext e n d e d t o 1: 1, f 5 7 will n ot b e all o w e d 


 
1 6  
§  I S O 4 0 0 
 
P h ot o gr a p h Pr oc e d ur e  
§  Pl ac e p ati e nt i nt o p ositi o n t h at will all o w m axi m u m st a bilit y; st a n di n g, sitti n g or “ h e a d-
cl a m p” p ositi o n. E m pl o yi n g vill a g e v ol u nt e er t o h el p is v er y us ef ul. 
§  T a k e p h ot o of p ati e nt I D 
§  I m a g e is br o u g ht i nt o f oc us b y c h a n gi n g t h e w or ki n g dist a nc e, n ot b y t ur ni n g t h e l e ns. 
T his  is  b ec a us e  t h e  l e ns  is  fix e d  i n  its  m a n u al  s etti n g.  T h e  w or ki n g  dist a nc e  is 
a p pr oxi m at el y 2 0 c m fr o m t h e e y e wit h o ur c urr e nt s etti n gs 
§  T a k e mi ni m u m of 2 p h ot os. If t h er e is a n y d o u bt of t h e q u alit y of t h e p h ot o w hil e t h e 
p ati e nt  is  i n  p ositi o n  it  is  b ett er  t o  c o nti n u e  t o  t a k e  m or e  p h ot o gr a p hs  b ef or e  t h e 
p ati e nt is all o w e d t o l e a v e. It is e as y t o d el et e p h ot os if t h e y ar e n ot n e e d e d. 
§  C h ec k  p h ot os  b ef or e  all o wi n g  c hil d  t o  l e a v e.  If  t h e y  ar e  n ot  acc e pt a bl e,  r e p e at 
pr oc e d ur e. O nl y st o p if t h e p ati e nt or g u ar di a n r e q u ests t h at w e st o p, or if is d e e m e d 
i m p ossi bl e,  e v e n  wit h  f urt h er  att e m pts.    N ot e  w e  h a v e  o bt ai n e d  > 9 5 %  acc e pt a bl e 
p h ot os i n pr e vi o us st u di es. 
§  If t h e c hil d c a n n ot b e p h ot o gr a p h e d f or s o m e r e as o n, it d o es n ot aff ect t h e eli gi bilit y of 
t h e  c hil d.  A  n ot ati o n  is  m a d e  o n  t h e  oc ul ar  f or m  of  w h y  t h e  p h ot o gr a p h  c a n n ot  b e 
t a k e n, b ut n o r e pl ac e m e nt is s o u g ht.  A g ai n, t his is ex p ect e d t o b e a r ar e e v e nt. 
 
P h ot o  Tr o u bl es h o oti n g G ui d e  
 
N ot e  all  c a m er a  s etti n gs  ar e  p er m a n e ntl y  e m b e d d e d  i n  e v er y  p h ot o  t h at  is  t a k e n  a n d  c a n  b e 
vi e w e d wit h t h e c a m er a or wit h a n y st a n d ar d c o m m erci al p h ot o- vi e wi n g s oft w ar e ( e. g., A d o b e 
P h ot os h o p; P h ot o m ec h a nic, Ni k o n Vi e w, C a n o n, etc.) 
§  P h ot os t o o D A R K, t o o LI G H T or O U T O F F O C U S 
o  C h ec k c a m er a s etti n gs: I S O 4 0 0, A p ert ur e pr ef err e d, +/- o n z er o, M a n u al s etti n g 
o n l e ns, fl as h el e v at e d; b att er y f ull y c h ar g e d 
§  If  all  p h ot os  ar e  aff ect e d  i n  t h e  s a m e  w a y  t h a n  it  is  m ost  li k el y  t h e  s etti n gs  o n  t h e 
c a m er a or l e ns 
o  C h a n g e c a m er a s etti n gs fr o m d ef a ult r ec o m m e n d ati o ns 
o  D ecr e as e F st o p t o li g ht e n, i ncr e as e t o d ar k e n 
o  C o ul d b e t a k e n d o w n t o 3 2 or l o w er b ut d e pt h of fi el d w ill b e l ess e n e d 
o  +/- c a n b e i ncr e as e d or d ecr e as e d t o c h a n g e ex p os ur e ( + t o li g ht e n; - t o d ar k e n) 
o  C h ec k t h at l e ns is o n M a n u al N O T A ut of oc us 
o  I S O- i ncr e as e t o li g ht e n p h ot o, d ecr e as e t o d ar k e n 
o  V er y hi g h I S O will pr o d uc e gr ai ni n ess 
o  M et er- tr y “s p ot” i nst e a d of “ p att er n 
o  H a v e a n ot h er p ers o n o bs er v e p h ot o gr a p h y i n r e al ti m e 
o  M a k e s ur e fl as h is n ot d e pr ess e d b y p h ot o gr a p h er or s o m e o n e els e w hil e p h ot o 
is  t a k e n.  ( T his  c o m m o nl y  h a p p e ns  if  p h ot o gr a p h er  h as  l o u p es).  Is  t h e  l e ns  w ell 
s u p p ort e d ? 
o  M a k e s ur e b att er y is f ull y c h ar g e d 


 
1 7  
§  If  first  p h ot os  i n  s eri es  ar e  acc e pt a bl e  a n d  t h e n  t h e y  gr a d u all y  b ec o m e  l ess  ex p os e d 
( d ar k er)  it  mi g ht  b e  b ec a us e  t h e  b att er y  is  gr a d u all y  l osi n g  p o w er  d uri n g  t h e  s essi o n. 
N ot e t h at t h e fl as h r e acti o n ti m e i ncr e as es as t h e b att er y p o w er d ecr e as es. 
§  O ut of f oc us 
o  F st o p m a y b e t o o l o w; m o v e m e nt artif act; fl as h d o es n’t w or k; l e ns is dirt y 
§  I m a g e n ot c e nt er e d 
o  M o v e m e nt  of  c hil d  or  c a m er a  ( h ol d  c hil d’s  h e a d  b et w e e n  k n e es  of  “ h el p er”; 
st a biliz e c a m er a wit h s ec o n d h a n d) 
§  R efl ecti o n artif act 
o  M o v e  c a m er a  sli g htl y  b et w e e n  first  a n d  s ec o n d  p h ot os  t o  ac hi e v e  diff er e nt 
a n gl e;  g e ntl y  d a b  c o nj u ncti v a  wit h  s w a b-  m ust  b e  d o n e  at  p eri p h er y  t o  a v oi d 
cr e ati n g i nfl a m m ati o n 
 
Gr a di n g of cli nic al c o nj u ncti v al p h ot o gr a p hs  
T hr e e ex p eri e nc e d o p ht h al m ol o gists will gr a d e t h e p h ot o gr a p hs at t h e c o ncl usi o n of t h e st u d y . 
Gr a d ers  will  gr a d e  e ac h  p h ot o gr a p h  i n  t h e  s et  f or  f ollicl es  a n d  i nfl a m m ati o n  acc or di n g  t o  t h e 
W H O si m plifi e d gr a di n g s yst e m.  
 
4. 3. 4 Cli nic al e v al u ati o n of t h e c o nj u ncti v a  
 
O n sit e cli nic al ex a mi n ati o n will b e p erf or m e d b y m e dic al st aff m e m b er pr e vi o usl y tr ai n e d. T h e 
ex a mi n er will us e t h e 2. 5 X m a g nif yi n g oc ul ar l o u p es ( Wils o n o p ht h al mics) t o ass ess t h e t ars al 
c o nj u ncti v a of t h e e v ert e d u p p er ri g ht e y eli d. T h e ex a mi n er will gr a d e t h e c o nj u ncti v a 
acc or di n g t o t h e W H O si m plifi e d Tr ac h o m a gr a di n g sc al e (s e e t a bl e b el o w). If n ec ess ar y, a 
h a n d- h el d t orc h li g ht will b e us e d b y t h e ex a mi n er f or ill u mi n ati o n of t h e c o nj u ncti v a.  T h e 
ex a mi n er t ells t h e Tr ac h o m a Gr a d e t o t h e t u b er, w h o t h e n r ec or ds t h e gr a d e o nt o t h e 
el ectr o nic d e vic e. Fi el d st aff will o nl y b e c ertifi e d t o gr a d e f or t h e st u d y if t h e y exc e e d s uffici e nt 
a gr e e m e nt wit h a n ex p ert tr ac h o m a gr a d er ( k a p p a ≥0. 6 o n a s eri es of 2 5 pict ur es) d uri n g t h e 
tr ai ni n g s essi o n. 
 
Defi niti o ns of t he W H O Si m plifie d Tr ac h o m a Gr a di n g Sc ale 
 
T F  ( Trac h o mat o us I nfla m mati o n – F ollic ular):  t he prese nce of fi ve or m ore f ollicles i n t he 
u p per tarsal c o nj u ncti va. 
TI  ( Trac h o mat o us I nfla m mati o n – I nte nse):  pr o n o u nce d i nfla m mat or y t hic ke ni n g of t he u p per 
tarsal c o nj u ncti va t hat o bsc ures m ore t ha n half of t he n or mal dee p tarsal vessels. 
T S  ( Trac h o mat o us Scarri n g):  t he prese nce of scarri n g i n t he tarsal c o nj u ncti va. 
T T  ( Trac h o mat o us Tric hiasis):  at least o ne e yelas h r u bs o n t he e ye ball. 
C O  ( C or neal O pacit y):  easil y visi ble c or neal o pacit y o ver t he p u pil. 
Cli nic ally active tr ac h o m a:  defi ne d as eit her T F or TI.  If W H O g ui deli nes rec o m me n d t h at T F 
al o ne is t he m ost a p pr o pri ate si g n t o f oll o w, t he n we will e asily be a ble t o re p ort t his.  
 
 
 


 
1 8  
5.  A N T H R O P O M E T R Y 
 
A nt hr o p o m etr y will b e c oll ect e d f or all c hil dr e n i n St u d y T w o at b as eli n e a n d 4 w e e ks aft er t h e 
c o m pl eti o n of t h e tr e at m e nt. W e will m e as ur e l e n gt h or h ei g ht, w ei g ht, a n d M U A C.  
 
M e as uri n g L e n gt h a n d H ei g ht 
A li g ht w ei g ht m e as uri n g b o ar d will b e us e d t o m e as ur e t h e p artici p a nt’s h ei g ht t o t h e n e ar est 
0. 1 c m. H ei g ht will b e ass ess e d wit h a S h orr B o ar d. D e p e n di n g o n a c hil d’s a g e a n d a bilit y t o 
st a n d, m e as ur e t h e c hil d’s l e n gt h or h ei g ht.   
 
L e n gt h: If a c hil d is l ess t h a n 2 y e ars ol d, or c a n n ot st a n d al o n e, m e as ur e r ec u m b e nt l e n gt h. A 
c hil d’s l e n gt h is m e as ur e d l yi n g d o w n (r ec u m b e nt).   
H ei g ht: If a c hil d is a bl e t o st a n d, w e will m e as ur e st a n di n g h ei g ht.  
 
Pr oc e d ur e  
If t h e c hil d h as br ai ds or h air or n a m e nts t h at will i nt erf er e wit h l e n gt h/ h ei g ht m e as ur e m e nts, 
r e m o v e t h e m if p ossi bl e.  C h ec k t h at a n y s a n d als, s h o es, or s oc ks h a v e als o b e e n r e m o v e d.  
W h et h er m e as uri n g l e n gt h or h ei g ht, t h e m ot h er/ g u ar di a n is n e e d e d t o h el p wit h 
m e as ur e m e nts a n d t o s o ot h e a n d c o mf ort t h e c hil d.  Ex pl ai n t o t h e m ot h er t h e r e as o ns f or t h e 
m e as ur e m e nts, a n d d escri b e t h e st e ps i n t h e pr oc e d ur e. A ns w er a n y q u esti o ns s h e mi g ht h a v e. 
S h o w h er a n d t ell h er h o w s h e c a n h el p y o u. Ex pl ai n t h at it is i m p ort a nt t o k e e p t h e c hil d still 
a n d c al m t o o bt ai n t h e b est m e as ur e m e nt.  
 
S h orr B o ar d S et -U p  
1)  R e m o v e S h orr B o ar d fr o m b a g. 
2)  St a n d t h e S h orr B o ar d u pri g ht.  Y o u c a n st e p o n t h e b as e of t h e b o ar d t o k e e p it st a bl e. 
3)  As y o u f ac e t h e b o ar d, t ur n t h e n o n-r e m o v a bl e b olt c o u nt ercl oc k wis e t o r el e as e t h e 
ext e nsi o n pi ec e.  N ot e: t h e b olt r e m ai ns att ac h e d t o t h e b ac k of t h e ext e nsi o n pi ec e – 
d o N O T r e m o v e it. 
4)  Sli d e t h e ext e nsi o n pi ec e i nt o t h e t o p e n d of t h e m ai n b o ar d a n d f ast e n t h e cl as p o n t h e 
b ac k of t h e b o ar d.  M a k e s ur e t h e cl as p is f ast e n e d pr o p erl y.   
5)  T h e a ut o-l oc k sli di n g h e a d/f o ot pi ec e is st or e d i n t h e b as e of t h e m ai n b o ar d a n d c a n b e 
m o v e d u p a n d d o w n t h e l e n gt h of t h e m e as uri n g b o ar d. It s h o ul d st a y i n pl ac e o n its 
o w n w h er e v er y o u p ositi o n it.   
6)  T h e m e as uri n g b o ar d m ust b e pl ac e d a g ai nst a fir m s urf ac e f or st a n di n g h ei g ht ( e. g. 
w all, t a bl e, tr e e, a g ai nst a v e hicl e, etc.). M a k e s ur e t h e b o ar d is st a bl e. If n ec ess ar y, 
pl ac e it e ms s uc h as s m all r oc ks u n d er n e at h t h e h ei g ht b o ar d t o st a biliz e it d uri n g t h e 
m e as ur e m e nt.  
7)  Cl e a n t h e e q ui p m e nt wit h alc o h ol s w a bs at t h e b e gi n ni n g of e ac h d a y.  
 
N ot e: w h e n y o u s et u p t h e b o ar d e ac h d a y, ex a mi n e e ac h of t h e pi ec es t o c h ec k f or d a m a g e. 
 
M e as ur e L e n gt h  
 


 
1 9  
C o v er t h e l e n gt h b o ar d wit h a c h uc k f or h y gi e n e a n d f or t h e b a b y’s c o mf ort. Ex pl ai n t o t h e 
m ot h er t h at s h e will n e e d t o pl ac e t h e b a b y o n t h e l e n gt h b o ar d a n d t h e n h el p t o h ol d t h e 
b a b y’s h e a d i n pl ac e w hil e t h e m e as ur e m e nt is t a k e n. S h o w h er w h er e t o st a n d w h e n pl aci n g 
t h e b a b y d o w n (i. e. o p p osit e y o u, o n t h e si d e of t h e l e n gt h b o ar d a w a y fr o m t h e t a p e).  Als o 
s h o w h er w h er e t o pl ac e t h e b a b y’s h e a d ( a g ai nst t h e fix e d h e a d b o ar d) s o t h at s h e c a n m o v e 
q uic kl y a n d s ur el y wit h o ut distr essi n g t h e b a b y. W h e n t h e m ot h er u n d erst a n ds y o ur 
i nstr ucti o ns a n d is r e a d y t o assist: As k h er t o l a y t h e c hil d o n his b ac k wit h his h e a d a g ai nst t h e 
fix e d h e a d b o ar d, c o m pr essi n g t h e h air.  
Q uic kl y p ositi o n t h e h e a d s o t h at a n i m a gi n ar y v ertic al li n e fr o m t h e e ar c a n al t o t h e l o w er 
b or d er of t h e e y e s oc k et is p er p e n dic ul ar t o t h e b o ar d.  ( T h e c hil d’s e y es s h o ul d b e l o o ki n g 
str ai g ht u p.) As k t h e m ot h er t o m o v e b e hi n d t h e h e a d b o ar d a n d h ol d t h e h e a d i n t his p ositi o n.   
S p e e d is i m p ort a nt. St a n di n g o n t h e si d e of t h e l e n gt h b o ar d w h er e y o u c a n s e e t h e m e as uri n g 
t a p e a n d m o v e t h e f o ot b o ar d: C h ec k t h at t h e c hil d li es str ai g ht al o n g t h e b o ar d a n d d o es n ot 
c h a n g e p ositi o n. S h o ul d ers s h o ul d t o uc h t h e b o ar d, a n d t h e s pi n e s h o ul d n ot b e arc h e d. As k t h e 
m ot h er t o i nf or m y o u if t h e c hil d arc h es t h e b ac k or m o v es o ut of p ositi o n. H ol d d o w n t h e 
c hil d’s l e gs wit h t h e o n e h a n d a n d m o v e t h e f o ot b o ar d wit h t h e ot h er. A p pl y g e ntl e pr ess ur e t o 
t h e k n e es t o str ai g ht e n t h e l e gs as f ar as t h e y c a n g o wit h o ut c a usi n g i nj ur y or distr ess.   
 
N ot e: it is n ot p ossi bl e t o str ai g ht e n t h e k n e es of n e w b or ns t o t h e s a m e d e gr e e as ol d er 
c hil dr e n. T h eir k n e es ar e fr a gil e a n d c o ul d b e e asil y i nj ur e d, s o a p pl y o nl y mi ni m u m pr ess ur e.   
 
If a c hil d is extr e m el y a git at e d a n d b ot h l e gs c a n n ot b e h el d i n p ositi o n, m e as ur e wit h o n e l e g i n 
p ositi o n. W hil e h ol di n g t h e k n e es, p ull t h e f o ot b o ar d a g ai nst t h e c hil d’s f e et. U p o n r e a di n g t h e 
m e as ur e m e nt, t h e ex a mi n er will cl e arl y c all o ut t h e n u m b er t o t h e r ec or d er. R ec or d t h e c hil d’s 
l e n gt h i n c e nti m et ers t o t h e l ast c o m pl et e d 0. 1 c m.  ( 1. 0 m m). K e e pi n g t h e c hil d i n pl ac e, 
r el e as e t h e sli di n g f o ot b o ar d, a n d pr e p ar e t o r e p e at t h e m e as ur e m e nt. R e- p ositi o n t h e c hil d f or 
a s ec o n d a n d t hir d m e as ur e m e nt.  
 
 
 
 
 


 
2 0  
M e as ur e St a n di n g H ei g ht 
 
E ns ur e t h at t h e h ei g ht b o ar d is o n l e v e l gr o u n d. W or ki n g wit h t h e m ot h er, a n d k n e eli n g i n 
or d er t o b e at t h e l e v el of t h e c hil d. H el p t h e c hil d st a n d o n t h e b as e b o ar d wit h t h e w ei g ht of 
t h e c hil d e v e nl y distri b ut e d o n b ot h f e et.  T h e h e els of t h e f e et ar e pl ac e d t o g et h er wit h b ot h 
h e els t o uc hi n g t h e b as e of t h e v ertic al b o ar d.  Pl ac e t h e f e et p oi nt e d sli g htl y o ut w ar d at a 6 0 
d e gr e e a n gl e. T h e b ac k of t h e h e a d, s h o ul d er bl a d es, b utt oc ks, c al v es, a n d h e els s h o ul d all 
t o uc h t h e v ertic al b o ar d. Ar ms s h o ul d h a n g fr e el y b y t h e si d es of t h e b o d y with t h e p al ms f aci n g 
t h e t hi g hs.  
 
N ot e: St a n di n g wit h all b o d y p arts t o uc hi n g t h e b o ar d m a y b e diffic ult f or s o m e c hil dr e n, i n 
w hic h c as e, h el p t h e c hil d t o st a n d o n t h e b o ar d wit h o n e or m or e c o nt act p oi nts t o uc hi n g t h e 
b o ar d. 
 
As k t h e m ot h er t o h ol d t h e c hil d’s k n e es a n d a n kl es t o h el p k e e p t h e l e gs str ai g ht a n d f e et fl at, 
wit h h e els a n d c al v es t o uc hi n g t h e v ertic al b o ar d. As k h er t o f oc us t h e c hil d’s att e nti o n, s o ot h e 
t h e c hil d as n e e d e d, a n d i nf or m y o u if t h e c hil d m o v es o ut of p ositi o n. P ositi o n t h e c hil d’s h e a d 
s o t h at a h oriz o nt al li n e fr o m t h e e ar c a n al t o t h e l o w er b or d er of t h e e y e s oc k et r u ns p ar all el 
t o t h e b as e b o ar d. As k t h e c hil d t o i n h al e d e e pl y a n d t o st a n d f ull y er ect wit h o ut alt eri n g t h e 
p ositi o n of t h e h e els.  If n ec ess ar y, p us h g e ntl y o n t h e b ell y t o h el p t h e c hil d st a n d t o f ull h ei g ht.  
Still k e e pi n g t h e h e a d i n p ositi o n, us e y o ur ot h er h a n d t o p ull d o w n t h e h e a d b o ar d t o r est 
fir ml y o n t o p of t h e h e a d a n d c o m pr ess t h e h air. U p o n r e a di n g t h e m e as ur e m e nt, t h e ex a mi n er 
will cl e arl y c all o ut t h e n u m b er t o t h e r ec or d er.  R ec or d t h e c hil d’s h ei g ht i n c e nti m et ers t o t h e 
l ast c o m pl et e d 0. 1 c m ( 1. 0 m m). K e e pi n g t h e c hil d i n pl ac e, r el e as e t h e sli di n g h e a d b o ar d, a n d 
pr e p ar e t o r e p e at t h e m e as ur e m e nt. R e- p ositi o n t h e c hil d f or a s ec o n d a n d t hir d m e as ur e m e nt.    


 
2 1  
 
 
 


 
2 2  
 
Dis m a ntli n g t h e S h orr B o ar d  
1)  St a n d t h e b o ar d u pri g ht: f ac e t h e b o ar d a n d st e p o n t h e b as e wit h o n e f o ot t o k e e p it 
st a bl e. 
2)  Sli d e t h e h e a d/f o ot pi ec e i nt o t h e b as e of t h e m ai n b o ar d. 
3)  R el e as e t h e cl as p o n t h e b ac k of t h e ext e nsi o n pi ec e a n d r e m o v e it.  P us h t h e cl as p FL A T 
a g ai nst t h e ext e nsi o n pi ec e. 
4)  T o att ac h t h e ext e nsi o n pi ec e t o t h e m ai n b o ar d, t ur n t h e fr o nt of t h e ext e nsi o n pi ec e 
i n w ar d a n d pl ac e it a g ai nst t h e fr o nt of t h e m ai n b o ar d.  M a k e s ur e t h at all si d es of t h e 
ext e nsi o n pi ec e ar e str ai g ht a n d i n li n e wit h t h e m ai n b o ar d. 
5)  P us h o n t h e b olt t h at is o n t h e b ac k of t h e ext e nsi o n pi ec e a n d scr e w it i nt o t h e m ai n 
b o ar d.   
6)  P ut t h e b o ar d b ac k i nsi d e of t h e c arr yi n g c as e f or st or a g e u ntil y o ur n ext us e.   
 
M e as uri n g W ei g ht 
 
T h e S E C A 8 7 4 sc al e will b e us e d t o w ei g h i nf a nts a n d c hil dr e n t o t h e n e ar est 0. 0 1 k g. I nf a nts 
a n d y o u n g c hil dr e n c a n als o b e w ei g h e d si m ult a n e o usl y wit h t h eir p ar e nt or g u ar di a n b y t h e 
u ni q u e “ m ot h er- b a b y” f u ncti o n ( p ar e nt or g u ar di a n is w ei g h e d a n d t h e n t h e i nf a nt or c h il d is 
w ei g h e d w hil e h el d b y t h e p ar e nt). Ex pl ai n t o t h e m ot h er t h at w e w a nt t o w ei g h h er c hil d t o 
s e e h o w h e or s h e is gr o wi n g.  If s h e h as a b a b y or a c hil d w h o is u n a bl e t o st a n d, s h e will h ol d 
t h e c hil d o n t h e sc al e.  If t h e c hil d is 2 y e ars or ol d er/c a n st a n d al o n e, t he c hil d will b e w ei g h e d 
al o n e. C hil dr e n s h o ul d b e w e ari n g o nl y li g ht cl ot hi n g, n o s h o es or s a n d als, n o h air or n a m e nts, 
a n d n o j e w elr y. Ex pl ai n t h at t h e c hil d n e e ds t o r e m o v e o ut er cl ot hi n g a n d s h o es/s a n d als i n 
or d er t o o bt ai n a n acc ur at e w ei g ht.  If t h e b a b y is w e ari n g a di a p er, t h e di a p er s h o ul d b e 
r e m o v e d.  If a n y h e a v y cl ot h es r e m ai n o n t h e c hil d, m a k e a n ot e i n t h e N ot es s ecti o n. 
 
S ec a 8 7 4 Sc al e  
1)  R e m o v e sc al e fr o m b a g. 
2)  B e s ur e t h at t h e sc al e is pl ac e d o n a fl at, h ar d, e v e n s urf ac e. All 4 l e gs of t h e sc al e s h o ul d 
m a k e c o nt act wit h t h e gr o u n d s urf ac e, wit h o ut w o b bli n g. It m a y b e h el pf ul t o pl ac e a 
pi ec e of pl y w o o d o n t h e gr o u n d u n d er n e at h t h e sc al e.  
3)  W h e n b att  a p p e ars i n t h e dis pl a y, y o u s h o ul d c h a n g e t h e b att eri es.  R e m o v e t h e ol d 
b att eri es a n d i ns ert 6 n e w b att eri es.   
 
T ur n t h e p o w er o n t h e sc al e w h e n y o u ar e r e a d y t o b e gi n w ei g hi n g. If t h e c hil d is u n a bl e t o 
st a n d o n t h e sc al e, y o u will us e t h e 2 i n 1 w ei g hi n g f u ncti o n (c all e d t ar e d w ei g hi n g). T h e 2 i n 1 
f u ncti o n e n a bl es t h e w ei g ht of b a bi es a n d s m all c hil dr e n t o b e d et er mi n e d w hil e a n a d ult h ol ds 
t h e m. I d e ntif y a s uit a bl e ar e a t h at is fl at f or h oriz o nt al pl ac e m e nt of t h e sc al e. Pr ess t h e st art 
k e y wit h n o l o a d o n t h e sc al e. T h e sc al e is r e a d y f or us e w h e n it s ets t o 0. 0 0. If n ec ess ar y, 
s witc h t h e w ei g ht dis pl a y t o K G: h ol d d o w n t h e 2 i n 1 k e y f or a b o ut 3 s ec o n ds. Pr ess t h e st art 
k e y wit h n o l o a d o n t h e sc al e. W ait u ntil t h e dis pl a y s h o ws 0. 0 0. 


 
2 3  
As k t h e a d ult t o r e m o v e his/ h er s h o es a n d st a n d i n t h e mi d dl e of t h e sc al e wit h o ut t h e c hil d. 
S/ h e s h o ul d r e m o v e a n y l o n g g ar m e nts, as t h es e c a n c o v er t h e dis pl a y a n d als o l e a d t o v ari a bl e 
m e as ur e m e nts. Aft er t h e a d ult’s w ei g ht a p p e ars o n t h e dis pl a y, t ell hi m/ h er t o r e m ai n st a n di n g 
o n t h e sc al e. Pr ess t h e 2 i n 1 k e y t o acti v at e t h e f u ncti o n. T h e sc al e st or es t h e w ei g ht of t h e 
a d ult a n d t h e dis pl a y r et ur ns t o z er o. W h e n 0. 0 0 a n d N E T a p p e ar i n t h e dis pl a y, h a n d t h e c hil d 
t o t h e a d ult.  T h e sc al e will d et er mi n e t h e w ei g ht of t h e c hil d.  O nc e t h e v al u e is st a bl e f or 
a b o ut 3 s ec o n ds, t h e w ei g ht is m e as ur e d.   
 
N ot e: If a n a d ult is v er y h e a v y ( e. g. m or e t h a n 1 0 0 k g) a n d t h e b a b y’s w ei g ht is r el ati v el y l o w 
( e. g. l ess t h a n 2. 5 k g), t h e b a b y’s w ei g ht m a y n ot r e gist er o n t h e sc al e.  I n s uc h c as es, h a v e a 
li g ht er p ers o n h ol d t h e b a b y o n t h e sc al e. 
 
T h e p ositi o n er will cl e arl y c all o ut t h e c hil d’s w ei g ht t o t h e r ec or d er. R ec or d t h e c hil d’s w ei g ht 
t o t h e n e ar est 0. 0 1 k g. R e p e at t h e m e as ur e m e nt 2 m or e ti m es. N ot e t h at o nl y t h e b a b y n e e ds 
t o r e m o v e d fr o m t h e sc al e; t h e a d ult s h o ul d r e m ai n o n t h e sc al e t h e e ntir e ti m e. T o t ur n off t h e 
2 i n 1 f u ncti o n, pr ess t h e 2 i n 1 k e y. T h e 2 i n 1 f u ncti o n r e m ai ns o n u ntil y o u pr ess t h e 2 i n 1 k e y 
a g ai n, or u ntil t h e sc al e s witc h es off a ut o m atic all y. If s e v er al c hil dr e n ar e t o b e w ei g h e d 
c o ns ec uti v el y wit h t h e s a m e a d ult h ol di n g t h e b a bi es, it is i m p ort a nt t h at t his p ers o n’s w ei g ht 
d o es n ot c h a n g e ( e. g. d u e t o a pi ec e of cl ot hi n g b ei n g r e m o v e d/ a d d e d). If t h e c hil d is a bl e t o 
st a n d o n t h e sc al e, y o u will w ei g h t h e c hil d al o n e. T al k wit h t h e c hil d a b o ut t h e n e e d t o st a n d 
still.  C o m m u nic at e wit h t h e c hil d i n a s e nsiti v e, n o n-fri g ht e ni n g w a y. Pr ess t h e st art k e y wit h n o 
l o a d o n t h e sc al e.  T h e sc al e is r e a d y f or us e w h e n it s ets t o 0. 0 0. As k t h e c hil d t o st a n d i n t h e 
mi d dl e of t h e sc al e. O nc e o n t h e sc al e, t h e c hil d m ust st a n d still. T h e H OL D f u ncti o n is 
a ut o m atic all y acti v at e d f or w ei g hts o v er 1. 5 k g/ 3. 3 l bs. T h e dis pl a y fl as h es u ntil a st a bl e w ei g ht 
h as b e e n m e as ur e d. T h e dis pl a y is t h e n fr oz e n u ntil t h e n ext w ei g hi n g o p er ati o n.   
 
N ot e: If t h e c hil d j u m ps o n t h e sc al e or w o n’t st a n d still, y o u will n e e d t o us e t h e t ar e d w ei g hi n g 
pr oc e d ur e i nst e a d.  
 
T h e p ositi o n er will cl e arl y c all o ut t h e c hil d’s w ei g ht t o t h e r ec or d er. R ec or d t h e c hil d’s w ei g ht 
t o t h e n e ar est 0. 0 1 k g. R e p e at t h e m e as ur e m e nt 2 m or e ti m es. H a v e t h e c hil d m o v e c o m pl et el y 
off t h e sc al e a n d t h e n st a n d a g ai n o n t h e sc al e. If n o f urt h er w ei g hi n g o p er ati o ns ar e 
p erf or m e d, t h e sc al e s witc h es off a ut o m atic all y aft er 2- 3 mi n ut es. 
 
Sc al e C ali br ati o n  
I n or d er t o m o nit or t h e c ali br ati o n of t h e sc al es o v er ti m e, e ac h t e a m will w ei g h a 5 k g t est 
w ei g ht at t h e b e gi n ni n g a n d e n d of t h e d a y. T his m e as ur e m e nt will t a k e pl ac e at t h e sit e of t h e 
sc al e st or a g e s o t h at t h e w ei g hts d o n ot n e e d t o b e c arri e d t o t h e fi el d. I n s o m e c as es, t h es e 
w ei g hts will b e k e pt i n a pr oj ect v e hicl e.  
 
 
 


 
2 4  
M e as uri n g Mi d- U p p er Ar m Circ u mf er e nc e ( M U A C) 
 
T h e c hil d’s M U A C will b e m e as ur e d o n e ti m e. M U A C m e as ur e m e nts will b e t a k e n at t h e 
mi d p oi nt of t h e l eft ar m b et w e e n t h e ti p of t h e s h o ul d er a n d t h e ti p of t h e el b o w usi n g n o n-
str etc h M U A C t a p es.  
 
First, fi n d t h e a p pr oxi m at e mi d p oi nt of t h e u p p er ar m. H a v e t h e c hil d st a n d 
u p str ai g ht wit h f e et t o g et h er, a n d t h e ri g ht ar m b e nt 9 0 d e gr e es at t h e 
el b o w, p al m f aci n g u p.  T h e ex a mi n er is p ositi o n e d b e hi n d t h e c hil d. If it is 
h el pf ul, m ar k t h e mi d p oi nt wit h a p er m a n e nt m ar k er. T o m e as ur e mi d- u p p er 
ar m circ u mf er e nc e, h a v e t h e c hil d st a n d u p str ai g ht wit h t h e ar ms r el ax e d at 
t h e si d es.  T h e ex a mi n er will st a n d f aci n g t h e c hil d’s ri g ht si d e.  T h e m e as uri n g t a p e is pl ac e d 
ar o u n d t h e u p p er ar m at t h e m ar k e d p oi nt. Wr a p t h e t a p e ar o u n d t h e ar m, p ulli n g it t o li e fl at 
a g ai nst t h e s urf ac e of t h e s ki n.  B e c ar ef ul n ot t o p ull t h e t a p e t o o ti g htl y (t o c o m pr ess t h e s ki n).   
 
T h e li n e f or r e a di n g m e as ur e m e nts is cl e arl y l a b el e d o n t h e M U A C 
t a p e ( R E A D (c m)). R e a d t h e n u m b er ali g n e d wit h t h e m e as ur e m e nt 
li n e o n t h e t a p e U p o n m e as uri n g, t h e ex a mi n er will cl e arl y c all o ut 
t h e n u m b er t o t h e r ec or d er.  R ec or d t o t h e n e ar est 0. 1 c m ( 0. 1 c m = 
1 m m). K e e pi n g t h e c hil d i n pl ac e, r el e as e t h e M U A C stri p. 
 
If t h e M U A C is < 1 1. 5c m r ef er t h e c hil d t o t h e n e ar est h e alt h c e nt er.  
 
 
6.  C O G NI TI V E D E V EL O P M E N T A S S E S S M E N T  
 
T h e D e v el o p m e nt al Mil est o n es C h ec klist ( D M C-III) will b e us e d t o e v al u at e t h e d e v el o p m e nt of 
m ot or a n d l a n g u a g e s kills i n 2 4 0 c hil dr e n at b as eli n e i n t his st u d y. T h e D M C-III i ncl u d es t hr e e 
s u bsc al es: gr oss m ot or, fi n e m ot or, a n d l a n g u a g e. T h e ori gi n al v ersi o n of t h e D M C-III w as 
d esi g n e d f or c hil dr e n a g e d 3 t o 2 4 m o nt hs. T h e u p d at e d D M C-III i ncl u d es a d diti o n al it e ms t o 
ass ess c hil dr e n a g e d 2 t o 8 y e ars. T h e D M C-III r e q uir es p ar e nt i nt er vi e w f or all it e ms a n d 
i ncl u d es o pti o n al dir ect o bs er v ati o n of t h e c hil d f or all it e ms. F or t his st u d y, d at a c oll ect ors will 
i ncl u d e b ot h p ar e nt i nt er vi e w a n d dir ect o bs er v ati o n f or all it e ms w h er e p ossi bl e. 
 
M at eri als f or o bs er v ati o n it e ms: 
•  P e ns  
•  W hit e p a p er 
•  A b all (c hil d-siz e f o ot b all/s occ er b all) 
•  A s m all t o y 
•  S m all gr ai ns i n a b ox 
•  A p ull -t o y 
•  A c ol or s h e et pri nt e d wit h b ox es of t h e f oll o wi n g c ol ors : bl u e, gr e e n, y ell o w, br o w n, 
p ur pl e, w hit e, gr e y, or a n g e, r e d, bl ac k, a n d pi n k 


 
2 5  
•  5 c oi ns   
 
Pr e p ari n g t h e ass ess m e nt s essi o n f or o bs er v ati o n it e ms:  
2. 1  B ef or e t h e ass ess m e nt s essi o n 
a)  C h ec k  t h at y o u h a v e all of t h e e q ui p m e nt  a n d r ec or d f or ms r e q uir e d usi n g t h e 
c h ec klist.  
 
2. 2  W h e n y o u arri v e at t h e c hil d’s h o us e 
a)  Gr e et  t h e m ot h er or c ar e gi v er acc o m p a n yi n g t h e c hil d. 
b)  I ntr o d uc e y o urs elf a n d o utli n e t h e p ur p os e of t h e visit  
c) A p pr o ac h  t h e c hil d f or t h e first ti m e i n t h e pr es e nc e of t h e m ot h er / c ar e gi v er  
d)  O bs er v e c ar ef ull y t h e c hil d’s r e acti o n t o y o urs elf a n d t h e sit u ati o n as it u nf ol ds.  
e)  C o nfir m  t h at t h e c hil d is i n g o o d h e alt h . D O N O T A S S E S S A C HIL D W H O I S 
U N W ELL . If t h e c hil d is sic k, c o n d uct t h e i nt er vi e w p orti o n of t h e visit a n d m a k e 
a n a p p oi nt m e nt t o r et ur n at a l at er d at e t o c o n d uct t h e o bs er v ati o n it e ms. 
 
2. 3  G ui d eli n es f or t h e t esti n g l oc ati o n 
a)  T h e t esti n g l oc ati o n s h o ul d b e a pl ac e w h er e t h er e is s h a d e, fl at gr o u n d, 
m axi m u m 
   
pri v ac y, a n d s p ac e f or m o v e m e nt. 
b)  T a k e ti m e t o s el ect t h e l ar g est, q ui et est l oc ati o n t h at all o ws y o u t o r u n ar o u n d. 
Y o u c a n sit 
i n o n e pl ac e a n d r u n ar o u n d els e w h er e.  
c) R e m o v e p ossi bl e  distr act ers  b ef or e st arti n g t o ass ess. 
d)  If p ossi bl e, p ositi o n t h e c hil d f aci n g a w all t o mi ni miz e distr acti o ns. 
e)  M a k e s ur e t h e c hil d a n d m ot h er ar e s ettl e d a n d c o mf ort a bl e. 
f) T a k e g o o d c ar e t o e ns ur e t h e s af et y of t h e c hil dr e n. L o o k o ut f or p ot e nti al 
h az ar ds. 
g)  If ot h ers g at h er t o w atc h, as k t h e m p olit el y t o l e a v e. Ex pl ai n t h at t h e c hil d mi g ht 
b e s h y if  
h e or s h e is b ei n g w atc h e d b y ot h ers. If n ec ess ar y, as k f or t h e m ot h er’s h el p t o 
dis p ers e t h e cr o w d. 
 
2. 4 S etti n g u p t h e e q ui p m e nt.  
a)  Arr a n g e  e q ui p m e nt w h er e it will b e e asil y acc essi bl e t o y o u.  
b)  E x pl ai n  t o t h e f a mil y m e m b er w h o acc o m p a ni es t h e c hil d t h eir r ol e. T o  
•  pr o vi d e c o mf ort t o t h eir c hil d b y t h eir pr es e nc e  
•  e nc o ur a g e t h e c hil d t o p artici p at e 
•  (if n ec ess ar y) t o h el p c o m m u nic at e t h e i nstr ucti o ns t o t h e c hil d  H el p 
t h e m t o u n d erst a n d t h e g a m e, b ut N O T t o assist t h e c hil d t o c o m pl et e 
t as ks.  
 
2. 5 S ettli n g t h e c hil d 
a)  Us e t h e n a m e t h e c hil d is c all e d b y t hr o u g h o ut t h e s essi o n. W h e n e v er y o u n e e d 


 
2 6  
t o g et t h e 
c hil d’s att e nti o n t o as k a q u esti o n or gi v e a n i nstr ucti o n, s a y his or h er n a m e.  
b)  R e - i ntr o d uc e y o urs elf t o t h e c hil d  
c) I n vit e t h e c hil d t o p artici p at e i n a n acti vit y. 
d)  E n g a g e  t h e c hil d wit h s o m e of t h e e q ui p m e nt/ acti viti es. T his m a y i ncl u d e a w ar m 
u p  
acti viti es, s e p ar at e fr o m t h e m ai n ass ess m e nt.  
e)  I n g e n er al, c hil dr e n r es p o n d w ell w h e n w e s mil e, ar e e nt h usi astic, pl a yf ul, a n d 
aff ecti o n at e. 
 
2. 6  G e n er al i nstr ucti o ns f or a d mi nist eri n g fi n e a n d gr oss m ot or it e ms: 
a)  First , d e m o nstr at e t h e t as k. 
b)  S ec o n d , h el p t h e c hil d t o d o t h e t as k. Gi v e s u p p ort w h er e n ec ess ar y u ntil y o u ar e 
c ert ai n t h at t h e c hil d c a n c o m pl et e t h e t as k u ns u p p ort e d. R e m o v e a n y s u p p ort 
pr o vi d e d i n st a g es.   
c) T hir d , t ell t h e c hil d t o d o t h e t as k al o n e, wit h o ut assist a nc e. 
 
2. 7  D uri n g ass ess m e nt. 
a.  C arr y o ut all acti viti es o n fl at a n d e v e n gr o u n d. 
b.  C hil d s h o ul d b e i n a cl e ar s p ac e a w a y fr o m f ur nit ur e / w alls. 
c. T h e i ncl usi o n of m ot h er a n d si bli n gs i n t h es e acti viti es oft e n ai ds a n d e nc o ur a g es t h e 
t ar g et c hil d t o f e el l ess i n hi bit e d. 
d.  Gi v e  pr ecis e a n d cl e ar i nstr ucti o ns t o t h e c hil d, f oll o wi n g m a n u al g ui d eli n es. 
e.  M o v e  fr o m o n e t as k t o t h e ot h er i n a s yst e m atic w a y.  
f. P a y att e nti o n  t o t h e c hil d at all ti m es.  
g.  Pr ais e  t h e c hil d f or eff ort, N O T s ucc ess i n a n it e m. 
h.  R et ur n ass ess m e nt m at eri al t o t h e b ox o n c o m pl eti o n of t h e it e m. T a k e o ut t h e 
m at eri al f or o n e it e m at a ti m e a n d r e pl ac e it w h e n y o u h a v e fi nis h e d. S o m eti m es y o u 
m a y t a k e o ut t h e m at eri al f or t h e n ext it e m b ef or e r e pl aci n g t h e m at eri al f or t h e 
pr e vi o us it e m, if t his is n ec ess ar y t o k e e p t h e c hil d’s att e nti o n. H o w e v er, i n t his c as e, 
r e pl ac e t h e m at eri al f or t h e pr e vi o us it e m i m m e di at el y aft er e n g a gi n g t h e c hil d’s 
att e nti o n wit h t h e n e w it e m. 
i. T a k e a br e a k  if t h e c hil d g ets tir e d or b ec o m es distr ess e d. I n g e n er al, it is a g o o d i d e a 
t o t a k e a br e a k at l e ast e v er y 3 0 mi n ut es. 
j. If n ec ess ar y, r e mi n d t h e acc o m p a n yi n g f a mil y m e m b er of t h eir r ol e i n t h e 
ass ess m e nt.  
 
D o N O T:  
1)  Cr o w d t h e ass ess m e nt ar e a wit h us e d it e ms, t his will distr act t h e c hild.  
2)  H urr y  t h e c hil d. 
 
2. 8  Str at e gi es f or h a n dli n g c hil dr e n w h o r ef us e 
a)  H a v e t h e m ot h er d e m o nstr at e t h e t as k a n d e nc o ur a g e t h e c hil d t o d o it 


 
2 7  
b)  I n v ol v e ot h er c hil dr e n w h o ar e ar o u n d, p er h a ps a n ol d er si bli n g. H a v e t h e ot h er c hil d d o 
it a n d s e e if t h e c hil d will d o t h e t as k al o n g wit h a n ot h er c hil d. 
c) Gi v e t h e c hil d a dri n k a n d a br e a k, l et t h e c hil d r est or pl a y f or a f e w mi n ut es 
d)  Pr o mis e t o gi v e t h e c hil d a s w e et if h e/s h e d o es t h e t as k 
 
2. 9  E n di n g t h e ass ess m e nt 
a)  F or m all y  e n d t h e s essi o n b y t h a n ki n g e v er y o n e. 
b)  B ef or e y o u l e a v e t h e h o us e  
i. R e a d  t hr o u g h y o ur f or m c ar ef ull y.  
ii. C o nfir m e ac h s ecti o n/ c h ec klist is fill e d f ull y.  
c) C h ec k all e q ui p m e nt is p ac k e d pr o p erl y.  ( Us e c h ec klist t o h el p y o u.) 
 
2. 1 0  
B ef or e t h e n ext s essi o n  
CL E A N A N D DI SI N F E C T T H E E Q UI P M E N T E V E R Y D A Y.  
        
A d mi nistr ati o n pr oc e d ur es  
I n e ac h s u bsc al e, t h e it e ms ar e arr a n g e d i n or d er fr o m t h e e arli er ac q uir e d s kill (f or ex a m pl e, 
sits s u p p ort e d) t o t h e l at er ac q uir e d s kill (f or ex a m pl e, sits al o n e o n t h e fl o or). T h e arr o ws i n 
t h e l eft- h a d m ar gi n s h o w t h e st art p oi nts f or t h e c hil d’s a g e. St art at t h e it e m a p pr o pri at e f or 
t h e c hil d’s a g e. If t h e c hil d sc or es 0, g o t o t h e pr e vi o us st art p oi nt. If t h e c hil d sc or es 1 or 2, 
c o nti n u e t o t h e n ext it e m. St o p aft er 4 c o ns ec uti v e sc or es of 0. R ef er t o t h e D M C-III M a n u al f or 
a d et ail e d d escri pti o n of h o w t o a d mi nist er e ac h it e m, i ncl u di n g pr o bi n g q u esti o ns t o us e if t h e 
i nt er vi e w e e h as diffic ult y u n d erst a n di n g t h e q u esti o n. T h e D M C-III fli pc h art i ncl u d es pict ur es, 
w hic h s h o ul d b e us e d i n c o nj u ncti o n wit h t h e q u esti o ns f or t h e t w o m ot or s u bsc al es. 
 
7.  MI C R O BI OL O GI C AL M E T H O D S    
 
P artici p a nt n a m es will n ot b e i ncl u d e d i n l a b or at or y r ec or ds a n d s a m pl es will b e d e i d e ntifi e d.  
T h er ef or e, l a b or at or y p ers o n n el will b e m as k e d, pr e v e nti n g i d e ntific ati o n of t h e i n di vi d u als 
i nf ect e d.  L a b r es ults will n ot b e a v ail a bl e f or w e e ks, if n ot m o nt hs. T h us, all i n di vi d u als will b e 
tr e at e d acc or di n g t o t h eir st u d y ar m, w h et h er or n ot t h eir l a b t ests r e v e al e vi d e nc e of i nf ecti o n. 
 
Pr e p ar ati o n of Z y m o D N A/ R N A S hi el d T M 
 
M a n uf act ur er’s Pr o d uct D escri pti o n 
D N A/ R N A S hi el d T M e ns ur es n ucl eic aci d st a bilit y d uri n g s a m pl e st or a g e/tr a ns p ort at a m bi e nt 
t e m p er at ur es, pr es er vi n g t h e g e n etic i nt e grit y a n d ex pr essi o n pr ofil es of s a m pl es. T h er e is n o 
n e e d f o r r efri g er ati o n or s p eci aliz e d e q ui p m e nt. D N A/ R N A S hi el dT M eff ecti v el y l ys es c ells a n d 
i n acti v at es n ucl e as es a n d i nf ecti o us a g e nts ( vir us), a n d it is c o m p ati bl e wit h v ari o us c oll ecti o n 
a n d st or a g e d e vic es ( v ac u u m t u b es, s w a bs, n as al, b ucc al, f ec al, etc.). 
 
M at eri als R e q uir e d  


 
2 8  
 
Pr o d uct  
S u p pli er  
C at al o g N o.  
D N A/ R N A S hi el d T M 
1 X s ol uti o n  
Z y m o R es e arc h C or p.  
R 1 1 0 0 -5 0 ( 5 0 ml)  
R 1 1 0 0 -2 5 0 ( 2 5 0 ml)  
2 ml L o o p Scr e w C a p Micr o T u b e, P C R P erf or m a nc e 
T est e d  
( D N A-, D N as e-, R N as e-, & P C R i n hi bit or-fr e e) 
S arst e dt I nc.  
7 2. 6 9 4. 4 1 6  
I ns ert f or L o o p Scr e w C a p, Vi ol et 
S arst e dt I nc.  
6 5. 7 1 3. 0 0 7  
 
Pr oc e d ur e  
1.  Pr e p ar e t u b es b y a d di n g vi ol et c a p i ns erts t o t h e t o p of e ac h t u b e (t h es e ar e f or c ol or 
c o di n g p ur p os es t o h el p disti n g uis h t h e D N A/ R N A S hi el d s ol uti o n fr o m ot h er m e di a 
b ei n g us e d i n t h e fi el d). 
2.  B ef or e ali q u oti n g t h e D N A/ R N A S hi el d, g e ntl y mix t h e s ol uti o n b y eit h er s wirli n g it or 
i n v erti n g t h e b ottl e a f e w ti m es (tr yi n g n ot t o cr e at e t o o m a n y b u b bl es) t o e ns ur e t h at 
t h e s ol uti o n is h o m o g e n o us. I n a cl e a n bi os af et y c a bi n et, us e st eril e t ec h ni q u e t o ali q u ot 
1 ml of t h e s ol uti o n i nt o e ac h 2 ml t u b e. 
3.  W h e n y o u h a v e fi nis h e d ali q u oti n g, m a k e s ur e t h at t h e c a p o n e ac h t u b e h as b e e n 
ti g ht e n e d t o pr e v e nt t u b es fr o m l e a ki n g d uri n g tr a ns p ort. 
 
St or a g e a n d S h elf Lif e  
St or e pr e p ar e d t u b es at r o o m t e m p er at ur e ( 2 0° C- 2 5° C) u ntil us e; d o n ot fr e ez e s ol uti o n pri or t o 
c oll ecti n g s a m pl es. Z y m o g u ar a nt e es t h e s h elf-lif e of D N A/ R N A S hi el d T M  u p t o 1 y e ar fr o m t h e 
d at e of r ec ei pt. T h er ef or e, if t h e s ol uti o n is ali q u ot e d ri g ht a w a y, t h e n pr e p ar e d t u b es s h o ul d 
b e g o o d f or 1 y e ar. T h e s h elf-lif e m a y b e l o n g er, d e p e n di n g o n st or a g e c o n diti o ns. 
 
M et h o ds f or R esist a nc e T esti n g 
 
Is ol ati o n & I d e ntific ati o n of Or g a nis ms Usi n g Micr o bi ol o gic al T ec h ni q u es 
 
S. p n e u m o ni a e will b e is ol at e d fr o m n as o p h ar y n g e al s w a bs usi n g bl o o d a n d str e p s el ect pl at e, 
c h oc ol at e, a n d c h oc ol at e wit h b acitr aci n m e di a. W e will i d e ntif y or g a nis ms usi n g gr a m st ai n. 
St a n d ar d micr o bi ol o gic al h a n d b o o ks s h o ul d b e r ef er e nc e d f or m or e d et ail e d i nf or m ati o n.  
 
L a b or at or y R es ults R e p orti n g 
 
All l a b r es ults will b e k e pt i n c o m p ut er fil es as w ell as i n h ar d-c o p y f or m b y t h e Micr o bi ol o gist. 
 
D escri pti o n of I d e ntific ati o n T ests:  


 
2 9  
W e will f oll o w t h e st a n d ar d q u alit y c o ntr ol m et h o ds alr e a d y i n pl ac e at t h e l a b or at or y. F or 
ex a m pl e, l a b st aff will p erf or m p ositi v e a n d n e g ati v e gr o wt h c o ntr ols f or all m e di a, a n d p ositi v e 
c o ntr ols f or all st ai ns a n d i d e ntific ati o n t ests at a pr e- d et er mi n e d sc h e d ul e. 
 
C at al as e T est:  
A f e w dr o ps of 3 % h y dr o g e n p er oxi d e ar e a d d e d t o c ol o ni es r e m o v e d fr o m a g ar a n d s m e ar e d 
o nt o a cl e a n gl ass sli d e, a n d i m m e di at el y o bs er v e d f or t h e r el e as e of ox y g e n or “ b u b bli n g.” 
B u b bli n g is i nt er pr et e d as t h e pr es e nc e of c at al as e. L ac k of b u b bli n g is a n e g ati v e t est. 
 
T u b e C o a g ul a s e: 
C o a g ul as e pl as m a is i n oc ul at e d wit h a n is ol at e d st a p h yl oc occ al c ol o n y a n d i nc u b at e d f or u p t o 
2 4 h o urs at 3 7° C. A n y d e gr e e of cl ot f or m ati o n is a p ositi v e r e acti o n a n d n o cl ot is a n e g ati v e 
r e acti o n. 
 
L at e x A g gl uti n ati o n: 
A f e w st a p h yl oc occ al c ol o ni es ar e mix e d wit h t h e l at ex a g gl uti n ati o n r e a g e nt a n d o bs er v e d f or 
a g gl uti n ati o n. A p ositi v e r e acti o n is a g gl uti n ati o n of t h e l at ex p articl es, w hil e n o a g gl uti n ati o n is 
a n e g ati v e r e acti o n. 
 
O pt oc hi n T est:  
A n o pt oc hi n disc is pl ac e d o nt o a bl o o d a g ar s u bc ult ur e d pl at e t h at h as b e e n i n oc ul at e d wit h a 
p ur e c ult ur e of t h e t est or g a nis m. Pl at es ar e i nc u b at e d f or 2 4 h o urs at 3 5° C i n C O 2. If a z o n e of 
i n hi biti o n of 1 4 m m or m or e is f or m e d, t h e n it’s S. p n e u m o ni a e . 
 
D es o x yc h ol at e ( 1 0 %) T est: 
A dr o p of r e a g e nt is a d d e d t o a w ell-is ol at e d c ol o n y o n a bl o o d a g ar pl at e. C ol o ni es of S. 
p n e u m o ni a e  will disi nt e gr at e aft er 3 0 mi n ut es. 
 
S at elliti n g Gr o wt h o n Bl o o d A g ar: 
C ol o ni es r es e m bli n g H a e m o p hil us ar e s u bc ult ur e d t o a n o n-s el ecti v e bl o o d a g ar pl at e a n d cr oss 
str e a k e d wit h C o a g ul as e n e g ati v e St a p h yl oc occ us. Pl at es ar e i nc u b at e d i n C O 2 o v er ni g ht a n d 
ex a mi n e d f or s at ellit e gr o wt h ar o u n d St a p h yl oc occ us . 
 
Micr o bi ol o gic al R esist a nc e T esti n g f or S w a bs 
R esist a nc e t esti n g will b e p erf or m e d usi n g t h e Kir b y B a u er disc diff usi o n ass a y or E-t est stri ps 
(t o d et er mi n e MI C’s). 
 
Q u alit y C o ntr ol 
E. c oli  A T C C 2 5 9 2 2, S. p n e u m o ni a e  A T C C 4 9 6 1 9, H. i nfl u e nz a e  A T C C 4 9 2 4 7, S. a ur e us  A T C C 
2 5 9 2 3 a n d 2 9 2 1 3 ar e us e d f or q u alit y c o ntr ol t esti n g of a nti bi otic discs a n d E-t est stri ps. 
 
Kir b y B a u er Ass a y 
A  s us p e nsi o n of t h e is ol at e is pr e p ar e d a n d t ur bi dit y a dj ust e d u ntil it r e ac h es t h e 0. 5 Mc F arl a n d 
st a n d ar d. T h e s us p e nsi o n is us e d t o i n oc ul at e t h e a p pr o pri at e s usc e pti bilit y a g ar pl at e f or 


 
3 0  
c o nfl u e nt gr o wt h. T h e pl at e is all o w e d t o dr y. A nti micr o bi al dis ks ar e pl ac e d o n t h e a g ar pl at es 
wit h st eril e f orc e ps or t w e ez ers; dis ks m a y n ot b e m o v e d o nc e t h e y h a v e t o uc h e d t h e pl at es. 
Aft er t h e dis ks ar e pl ac e d o n t h e pl at e, t h e pl at es ar e i n v ert e d a n d i nc u b at e d at 3 5° C f or 1 6 t o 
1 8 h o urs. Aft er i nc u b ati o n, t h e di a m et er of t h e z o n es of c o m pl et e i n hi biti o n (i ncl u di n g t h e 
di a m et er of t h e dis k) ( Fi g ur e 2) ar e m e as ur e d a n d r ec or d e d i n milli m et ers. T h e m e as ur e m e nts 
c a n b e m a d e wit h a r ul er or sli di n g c ali p er o n t h e u n d ers urf ac e of t h e pl at e wit h o ut o p e ni n g t h e 
li d. T h e z o n es of gr o wt h i n hi biti o n will b e c o m p ar e d wit h t h e z o n e-siz e i nt er pr et ati v e t a bl e a n d 
r ec or d e d as s usc e pti bl e, i nt er m e di at e, or r esist a nt t o e ac h dr u g t est e d acc or di n g t o CL SI 
g ui d eli n es (s e e A p p e n dix).  
 
 
Fi g ur e 2.  R es ults of t h e dis k diff usi o n ass a y. T his S hi g ell a  is ol at e is r esist a nt t o tri m et h o pri m-
s ulf a m et h ox az ol e a n d is gr o wi n g u p t o t h e dis k ( S X T), t h e z o n e of w hic h is r ec or d e d as 6 m m. 
 
E- t est stri ps. 
I n a d diti o n, azit hr o m yci n Mi ni m u m I n hi bit or y C o nc e ntr ati o ns ( MI C) ar e d et er mi n e d usi n g E-t est 
stri ps. A  s us p e nsi o n of t h e is ol at e is pr e p ar e d a n d t ur bi dit y a dj ust e d u ntil it r e ac h es t h e 0. 5 
Mc F arl a n d st a n d ar d. T h e s us p e nsi o n is us e d t o i n oc ul at e t h e a p pr o pri at e s usc e pti bilit y a g ar 
pl at e f or c o nfl u e nt gr o wt h. T h e pl at e is all o w e d t o dr y. E-t est stri ps ar e pl ac e d o n t h e a g ar 
pl at es wit h st eril e f orc e ps or t w e ez ers; stri ps m a y n ot b e m o v e d o nc e t h e y h a v e t o uc h e d t h e 
pl at es. Aft er t h e stri ps ar e pl ac e d o n t h e pl at e, t h e pl at es ar e i n v ert e d a n d i nc u b at e d at 3 5° C f or 
2 0  t o 2 4 h o urs. T h e MI C is r e a d w h er e t h e elli ps e i nt ers ects t h e MI C sc al e o n t h e stri p. T h e MI C 
will b e c o m p ar e d wit h t h e i nt er pr et ati v e t a bl e acc or di n g t o CL SI g ui d eli n es (s e e A p p e n dix).  
 
8.  P R O T E C TI O N O F H U M A N S U BJ E C T S 
 
B ef or e t h e st u d y b e gi ns, e ac h sit e will o bt ai n f or m al et hic al a p pr o v al fr o m t h eir r es p ecti v e 
n ati o n al et hics c o m mitt e e. I n a d diti o n, l oc al st aff will a p pr o ac h c o m m u nit y l e a d ers t o d escri b e 
t h e st u d y a n d a ns w er a n y q u esti o ns. St u d y st aff will pr oc e e d o nl y if l oc al l e a d ers hi p c o ns e nts t o 
p artici p at e. F or St u d y O n e, w e will o bt ai n v er b al c o ns e nt fr o m a p ar e nt or g u ar di a n of all st u d y 
p artici p a nts. F or St u d y T w o, w e will o bt ai n writt e n c o ns e nt fr o m a p ar e nt or g u ar di a n f or all 


 
3 1  
st u d y acti viti es, i ncl u di n g tr e at m e nt a n d ex a mi n ati o ns. C hil dr e n will o nl y b e i ncl u d e d i n t h e 
st u d y f oll o wi n g r ec ei pt of c o ns e nt fr o m t h e p ar e nt or g u ar di a n. If, at a n y ti m e, a p ar e nt or 
g u ar di a n el ects t o wit h dr a w a f a mil y m e m b er fr o m t h e st u d y, t h e y will b e fr e e t o d o s o. 
C hil dr e n wit h w asti n g or st u nti n g will b e r ef err e d f or a p pr o pri at e tr e at m e nt b y tr ai n e d st u d y 
p ers o n n el t o t h e n e ar est h e alt h c e nt er. 
 
Ris ks a n d B e n efits of St u d y Pr oc e d ur es 
 
S w a b bi n g Pr oc e d ur es  
T h er e ar e mi ni m al ris ks t o t h e p artici p a nt w h o r ec ei v es n as o p h ar y n g e al, a n d n ar es s w a b bi n g. 
P artici p a nts m a y ex p eri e nc e s o m e t e m p or ar y disc o mf ort, b ut t h e s w a b bi n g i n v ol v es mi ni m al 
ris k. A n y a d v ers e eff ects, s uc h as n os e- bl e e ds, will b e tr e at e d i m m e di at el y b y t h e ex a mi n ers. 
Ot h er h e alt h c ar e will b e pr o vi d e d at n o c ost t o t h e st u d y p artici p a nt if n ec ess ar y t o a d dr ess a 
st u d y-r el at e d a d v ers e h e alt h e v e nt. 
 
R ect al s w a bs a n d st o ol s a m pl es: R ect al s w a bs ar e a si m pl e pr oc e d ur e wit h v er y mi ni m al ris k.  
St o ol s a m pl e c oll ecti o n i n a n o n-i n v asi v e pr oc e d ur e wit h n o ass oci at e d ris ks.  
 
A nt hr o p o m etric M e as ur e m e nts  
T h er e ar e mi ni m al ris ks ass oci at e d wit h t h e m e as uri n g b o ar d, sc al e, or M U A C t a p es asi d e fr o m 
a nxi et y d uri n g t h e m e as ur e m e nts. Ex a mi n ers will d o t h eir b est t o e ns ur e t h at t h e 
p ar e nt/ g u ar di a n of t h e c hil d u n d erst a n ds t h e pr oc ess of ass essi n g a nt hr o p o m etric 
m e as ur e m e nts.  T h e ex a mi n ers will att e m pt t o mi ni miz e disc o mf ort f or all st u d y p artici p a nts 
b ef or e, d uri n g, a n d aft er t h e m e as ur e m e nts ar e t a k e n. C hil dr e n wit h w asti n g or st u nti n g will b e 
r ef err e d f or a p pr o pri at e tr e at m e nt at t h e n e ar est h e alt h c e nt er. 
 
9.  A P P E N DI C E S 
 


 
3 2  
 
1 0.  R E F E R E N C E S 
 
1  
W a n g H, B h utt a Z A, C o at es M M, et al.  Gl o b al, r e gi o n al, n ati o n al, a n d s el ect e d s u b n ati o n al 
l e v els of still birt hs, n e o n at al, i nf a nt, a n d u n d er - 5 m ort alit y, 1 9 8 0 – 2 0 1 5: a s yst e m atic 
a n al ysis f or t h e Gl o b al B ur d e n of Dis e as e St u d y 2 0 1 5. T h e L a nc et  2 0 1 6; 3 8 8 : 1 7 2 5– 7 4.  
2  
M ull er O, G ar e n n e M, K o u y at e B, B ec h er H. T h e ass oci ati o n b et w e e n pr ot ei n – e n er g y 
m al n utriti o n, m al ari a m or bi dit y a n d all-c a us e m ort alit y i n W est Afric a n c hil dr e n. Tr o pic al 
M e dici n e & I nt er n ati o n al H e alt h  2 0 0 3; 8 : 5 0 7– 1 1.  
3  
B ei ers m a n n C, B o u nt o g o M, Ti e n dr é b e o g o J, et al.  M al n utriti o n i n y o u n g c hil dr e n of r ur al 
B ur ki n a F as o: c o m p aris o n of s ur v e y d at a fr o m 1 9 9 9 wit h 2 0 0 9. Tr o pic al M e dici n e & 
I nt er n ati o n al H e alt h 2 0 1 2; 1 7 : 7 1 5– 2 1.  
4 
P orc o T C, G e br e T, A y el e B, et al.  Eff ect of M ass Distri b uti o n of Azit hr o m yci n f or Tr ac h o m a 
C o ntr ol o n O v er all M ort alit y i n Et hi o pi a n C hil dr e n: A R a n d o miz e d Tri al. J A M A 2 0 0 9; 3 0 2 : 
9 6 2 – 8.  
5  
G o u g h E K, M o o di e E E M, Pr e n d er g ast AJ, et al.  T h e i m p act of a nti bi otics o n gr o wt h i n 
c hil dr e n i n l o w an d mi d dl e i nc o m e c o u ntri es: s yst e m atic r e vi e w a n d m et a- a n al ysis of 
r a n d o mis e d c o ntr oll e d tri als. B MJ  2 0 1 4; 3 4 8 : g 2 2 6 7– 7.  
6  
Is a n a k a S, L a n g e n d orf C, B ert h é F, et al.  R o uti n e A m oxicilli n f or U nc o m plic at e d S e v er e 
Ac ut e M al n utriti o n i n C hil dr e n. N E n gl J M e d  20 1 6; 3 7 4 : 4 4 4– 5 3.  
7  
Tr e h a n I, G ol d b ac h H S, L a Gr o n e L N, et al.  A nti bi otics as P art of t h e M a n a g e m e nt of S e v er e 
Ac ut e M al n utriti o n. N E n gl J M e d  2 0 1 3; 3 6 8 : 4 2 5– 3 5.  
8  
B a n g A T, B a n g R A, T al e O, et al.  R e d ucti o n i n p n e u m o ni a m ort alit y a n d t ot al c hil d h o o d 
m ort alit y b y m e a ns of c o m m u nit y- b as e d i nt er v e nti o n tri al i n G a dc hir oli, I n di a. T h e L a nc et  
1 9 9 0; 3 3 6 : 2 0 1– 6.  
9  
O n a k p o y a IJ, H a y w ar d G, H e n e g h a n CJ. A nti bi otics f or pr e v e nti n g l o w er r es pir at or y tr act 
i nf ecti o ns i n hi g h-ris k c hil dr e n a g e d 1 2 y e ars a n d u n d er. C oc hr a n e D at a b as e Syst R ev  2 0 1 5; 
9 : C D 0 1 1 5 3 0. 
1 0   M el es e M, Al e m a y e h u W, L a k e w T, et al.  C o m p aris o n of A n n u al a n d Bi a n n u al M ass 
A nti bi otic A d mi nistr ati o n f or Eli mi n ati o n of I nf ecti o us Tr ac h o m a. J A M A 2 0 0 8; 2 9 9 : 7 7 8– 8 4.  
1 1   M el es e M, C hi d a m b ar a m J D, Al e m a y e h u W, et al.  F e asi bilit y of Eli mi n ati n g Oc ul ar 
C hl a m y di a tr ac h o m atis Wit h R e p e at M ass A nti bi otic Tr e at m e nts. J A M A 2 0 0 4; 2 9 2 : 7 2 1– 5.  
1 2   G e br e T, A y el e B, Z eri h u n M, et al.  C o m p aris o n of a n n u al v ers us t wic e- y e arl y m ass 
azit hr o m yci n tr e at m e nt f or h y p er e n d e mic tr ac h o m a i n Et hi o pi a: a cl ust er-r a n d o mis e d 
tri al. L a nc et 2 0 1 2; 3 7 9 : 1 4 3– 5 1.  


 
3 3  
1 3   H ar di n g- Esc h E M, Sill a h A, E d w ar ds T, et al.  M ass Tr e at m e nt wit h Azit hr o m yci n f or 
Tr ac h o m a: W h e n Is O n e R o u n d E n o u g h ? R es ults fr o m t h e P R E T Tri al i n T h e G a m bi a. PL o S 
N e gl Tr o p Dis  2 0 1 3;  7 : e 2 1 1 5– 1 2.  
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SAP_001.pdf: 
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Antibiotic Resistance and Microbiome in Children Aged 1-59 months in Nouna, Burkina Faso 
 
 
Manual of Operations and Procedures 
 
NCT number 03187834 
 
Version 1.1 June 2017 
 
Centre de Recherche en Santé de Nouna 
 
University of Heidelberg 
 
Francis I. Proctor Foundation, University of California, San Francisco 
 
 


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Antibiotic resistance and Microbiome in Children Aged 6-59 months in Nouna, Burkina Faso 
 
Statistical Analysis Plan, Version 1.0, June 15 2017 
 
Study Overview 
 
We will randomize 120 households containing 2 or more children aged 6-59 months to one of 
four study arms: 1) azithromycin, 2) amoxicillin, 3) cotrimoxazole, or 4) placebo. All children will 
receive 5 days of treatment. Full intervention details can be found in the Manual of Operations 
and Procedures. Rectal and nasopharyngeal swabs will be collected pre-treatment (baseline) 
and on Day 9 following the 5 days of treatment. Anthropometric indices (weight, height, and 
mid-upper arm circumference) will be collected at baseline and Day 35 (one month following 
treatment completion). 
 
Randomization 
 
The 120 households will be randomized in blocks of size 8 to one of the four treatment arms. In 
households randomized to treatment, all children aged 6-59 months of age will be treated, with 
the exception of one random child from within the household who will receive placebo. All 
children aged 6-59 months in households randomized to placebo will receive placebo. A 
random treated child from antibiotic arm households will be selected as the sentinel child for 
outcome assessment, and the untreated child from antibiotic arm households will also receive 
outcome assessment. Two children will be randomly selected from placebo households for 
outcome assessment. 
 
Primary Outcome 
 
We will compare diversity across arms at Day 9 and between time points. The primary outcome 
will be Simpson’s index of alpha diversity. We will compare the mean Simpson’s index between 
treatment arms with a t-test. All P-values will be calculated via permutation test. 
 
Secondary Outcomes 
 
As a secondary outcome, we will compare Shannon’s index (L1 norm) between time points 
using a t-test. To compare if treatment significantly explains difference between the four 
treatment groups, we will compare Simpson’s index (L1-norm) and Shannon’s index (L2-norm) 
distances using PERMANOVA on bacterial reads. 
 
To determine if there are differences in nutritional outcomes across treatment arms one month 
following treatment, we will calculate weight-for-height (WHZ), weight-for-age (WAZ), and 
height-for-age (HAZ) Z-scores based on 2006 WHO standards. We will compare mean WHZ, 
WAZ, HAZ, and mid-upper-arm circumference (MUAC) across treatment arms as continuous 
variables adjusting for baseline anthropometric values using an ANCOVA. We will also compare 


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the prevalence of stunting (HAZ <-2 SD) and moderate (WHZ <-2 SD) and severe (WHZ <-3 SD) 
acute malnutrition across study arms with a logistic regression model.