High prevalence of selected viruses and parasites and their predictors in Malawian children

Epidemiology and In ec ion camb idge.o g/hyg O iginal Pape Ci e his a icle: Fan Y-M, Oika inen S, Leh o K-M, Nu minen N, Juu i R, Mangani C, Male a K, Hyö y H, Asho n P (2019). High p e alence o selec ed i uses and pa asi es and hei p edic o s in Malawian child en. Epidemiology and In ec ion 147,e90,1–9. h ps://doi.o g/ 10.1017/S0950268819000025 Recei ed: 6 No embe 2017 Re ised: 16 Decembe 2018 Accep ed: 1 Janua y 2019 Key wo ds: In ec ion; pa asi e; p edic o ; p e alence; i us Au ho o co espondence: Y.-M. Fan, E-mail: [email p o ec ed] © The Au ho (s) 2019. This is an Open Access a icle, dis ibu ed unde he e ms o he C ea i e Commons A ibu ion licence (h p:// c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. High p e alence o selec ed i uses and pa asi es and hei p edic o s in Malawian child en Y.-M. Fan1, S. Oika inen2, K.-M. Leh o1, N. Nu minen2, R. Juu i3, C. Mangani1,4, K. Male a4, H. Hyö y2and P. Asho n1,5 1 Cen e o Child Heal h Resea ch, Tampe e Uni e si y, Facul y o Medicine and Heal h Technology and Tampe e Uni e si y Hospi al, Tampe e, Finland; 2 Depa men o Vi ology, Facul y o Medicine and Heal h Technology, Tampe e Uni e si y, Tampe e, Finland; 3 EPID Resea ch Oy, Espoo, Finland; 4 College o Medicine, Uni e si y o Malawi, Blan y e, Malawi and 5 Depa men o Paedia ics, Tampe e Uni e si y Hospi al, Tampe e, Finland Abs ac En e ic pa hogens ha e been ela ed o child unde nu i ion. Whe eas he e a e lo s o da a on en e ic bac e ial mic obio a and in ec ions, much less is known abou he incidence o p e a- lence o in es inal colonisa ion wi h i uses o impo an pa asi ic species. This s udy assessed he p esence o selec ed i uses and pa asi es in s ools o 469, 354, 468 Malawian child en a 6, 12 and 18 mon hs. We also assessed en i onmen al p edic o s o he p esence o i uses and pa asi es among 6-mon h in an s. Mic obial p esence was documen ed using eal- ime poly- me ase chain eac ion (PCR). En e o i uses we e iden i ied in 68%, 80% and 81% o he s ool samples a 6, 12 and 18 mon hs child en, hino i us in 28%, 18% and 31%, no o i us in 24%, 22% and 16%, pa echo i us in 23%, 17% and 17%, o a i us in 3%, 1% and 0.6%, Gia dia lamblia in 9.6%, 23.5% and 26%, and C yp ospo idium (spp.) in 6%, 8% and 2% o he 6, 12 and 18 mon hs s ool samples. D y season (May–Oc obe ) was associa ed wi h a low in ec- ion a e o en e o i us, no o i us and C yp ospo idium (spp.). Highe a he ’s educa ion le el, less numbe o pe son in he household and highe sani a ion we e associa ed wi h a low in ec ion a e o en e o i us, no o i us and o a i us, espec i ely. The esul s sugges ha he p e alence o asymp oma ic i al and pa asi ic in ec ions is high among Malawian chil- d en and ha he amily’s li ing condi ions and seasonali y in luence he a e o in ec ions. In oduc ion S un ing, de ined as child en’s leng h- o -age o heigh - o -age, is mo e han wo s anda d de ia ions below he Wo ld Heal h O ganisa ion (WHO) Child G ow h S anda d median. Globally, i is es ima ed ha 165 million child en younge han 5 yea s we e a ec ed by s un - ing in 2011. The p e alence has been dec easing du ing he pas decades, bu is s ill high in Sub-Saha an A ica and Sou h Asia (36% and 27%) [1]. In childhood, s un ing is associa ed wi h an inc eased isk o mo bidi y and mo ali y as well as delayed men al and mo o de el- opmen [1]. Impai ed ea ly childhood unde nu i ion in u n has been associa ed wi h lowe educa ional achie emen , lowe adul p oduc i i y and ea ning po en ial [2]. The mos common cause o s un ing is belie ed o be esul s o inadequa e die a y in ake, equen in ec ions o combina ion o bo h. In ecen yea s, en i onmen al en e opa hy, also known as en i onmen al en e ic dys unc ion (EED), has been s udied as ano he possible cause [3,4]. This condi ion is cha ac e ised by in es inal in lamma ion, di use illous a ophy and impai ed nu ien abso p ion and g ow h. I is belie ed o ha e an in ec ious o igin [5], bu nei he an ibio ic no p obio ic ea men seems o modi y i s cou se [6,7]. Vi al and pa a- si ic in ec ions would no be a ec ed by an ibac e ial in e en ions bu hey do cause in es inal in lamma ion esponse [8,9]. Fu he mo e, whole- ansc ip ome analysis om Malawian chil- d en wi h a ying s a es o EED showed ha 57% ansc ip s wi h inc eased exp ession in EED we e epo ed o espond o i al in ec ion [10]. Vi al o pa asi ic in ec ions o he gas oin es- inal ac a e also o en common bu asymp oma ic [11–18]; o ins ance, in a pilo s udy o 44 6-mon h-old in an s in u al Malawi, we de ec ed en e o i al RNA in 50% o he es ed s ool samples [19]. The s ool samples we e collec ed as pa o he same in e en ion ial as he cu en s udy. Gi en all hese indings, i seems plausible ha asymp oma ic in es inal i al o pa asi ic in ec ions would be con ibu ing o EED. To ge a mo e comp ehensi e pic u e o he equency o in es inal i al o pa asi ic in ec- ions among child en in a low-income se ing, we de e mined he p e alence o i e i uses- en e o i us, no o i us, hino i us, pa echo i us, o a i us and wo pa asi es –Gia dia lamblia and C yp ospo idium species in s ools om 6, 12 and 18 mon hs Malawian child en. We ocused on hese pa hogens because hey a e widely sp ead and e lec he hygienic condi ions h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. whe e he child en a e li ing. As an a emp o iden i y po en ial a ge s o p e en i e in e en ions, we also sough o iden i y majo en i onmen al o socio-demog aphic p edic o s o being in ec ed wi h selec ed i uses o pa asi es among 6-mon h in an s. Me hods S udy design, s udy subjec s and e hics This s udy was a c oss-sec ional s udy using da a and s ool sam- ples ha we e collec ed om u al Malawi. The 6-mon h in an s we e ec ui ed o a clinical die a y supplemen a ion ial be ween Janua y 2008 and May 2009, a ha poin we examined he in an s, in e iewed hei gua dians and collec ed and s o ed a s ool sample om hem. The in an s we e ollowed up o 12 mon hs and s ool samples we e also collec ed when hey we e 12- and 18-mon h-old. De ails o his ial, designa ed Lungwena Child Nu i ion In e en ion S udy (LCNI-5), ha e been desc ibed elsewhe e [20]. In b ie , he s udy popula ion comp ised 840 6-mon h-old in an s, who we e en olled o a clinical ial assessing he e ec o die a y supplemen a ion wi h lipid-based nu ien supplemen s o 12 mon hs on ea ly childhood g ow h ( egis a ion ID: NCT00524446, a h ps://clinical ials.go /). This ial was con- duc ed in Lungwena and Malindi, wo u al Malawian communi- ies. Malindi is abou 17 km om Mangochi own and had on a e age be e access o elec ici y, clean wa e , sani a ion and mis- siona y hospi al and a mo e educa ed popula ion han Lungwena, a mo e u al si e abou 32 km om he own. Bo h a eas a e along he sho es o Lake Malawi, Malawi. (Fig. 1). The ial adhe ed o he p inciples o he Decla a ion o Helsinki and egula o y guidelines in Malawi. W i en in o med consen was ob ained om he pa icipan s’gua dians and he ial p o ocol was e iewed and app o ed by he College o Medicine esea ch and e hics commi ee (Uni e si y o Malawi) and he e hical commi ee o he Pi kanmaa Hospi al Dis ic , Finland. Specimen collec ion, da a collec ion and s o age All s ool samples we e collec ed wi hin 14 days a e he en ol- men . The gua dians o he ial pa icipan s we e gi en a con- aine o collec he s ool samples om he pa icipan s, and hey b ough he sample o he heal h cen e a e wa ds. We col- lec ed da a wi h pic u e calenda eco ded by ca e ake (gua d- ian). Th oughou he s udy, he gua dians we e asked o eco d on a daily basis he p esence o absence o illness symp oms in pic u e calenda s ha we e p o ided e e y 2 weeks. The calenda had sepa a e ows o di e en days up o 2 weeks and sepa a e columns o he ollowing symp oms: (1) e e ; (2) cough; (3) dia hoea (≥3 s ools/d); and (4) o he . The i s 3 symp om col- umns had pic u es desc ibing hem o assis he gua dians in iden i ying he co ec a ea o eco d he in o ma ion. The gua d- ian epo s on he calenda s did no include he eco ding o em- pe a u e o e e o he numbe o s ools o dia hoea. The eco ded in o ma ion was e iewed and c oss-checked by ield- wo ke s a each 2 weekly ood-deli e y isi o comple eness. Those pa icipan s ca ying he pa hogens bu did no ha e illness symp oms and looked ‘heal hy’ om hei ca e ake s’ iews com- p ised ou s udy subjec s. F om he in o ma ion we collec ed a en olmen , 31 child en had dia hoea in he las 14 days, 22 du - ing he las 7 days and 11 in he las 2 days. The pa hogen in ec ions we e iden i ied by PCR om s ool samples collec ed a 6, 12 and 18 mon hs. Time be ween de eca ion and s ool eezing was ypically 1– 3 h, maximally 6 h. Abou 1 g s ool sample was aliquo ed by a labo a o y echnician o a sc ew cap collec ion ube. The samples we e s o ed o a maximum o 1 week a −20 °C in he s udy si e, 2–4 yea s a −40 °C a a cen al labo a o y in Mangochi and 2–7 mon hs a −80 °C a he Uni e si y o Tampe e whe e he analysis was done. The sample shipmen om Malawi o Tampe e ook place in d y ice. Sample p epa a ions, nucleic acid ex ac ion and eal- ime PCR F ozen s ool samples we e hawed and p ocessed in o 10% (w/ ) suspension in HANKS medium con aining 0.2% BSA. Nucleic acids we e isola ed using QIAamp Vi al RNA Mini Ki (Qiagen, Hilden, Ge many) om he 10% aecal suspension and eal- ime PCR was used o de ec en e o i uses, hino i uses, no o i uses, pa echo i uses and o a i uses [21,22] as well as Gia dia lamblia and C yp ospo idium (spp.) pa asi es as p e iously epo ed [23]. The p ime s and p obes used o PCR we e shown in supplemen- a y Table S1. Fo each pa hogen, we an h ee imes PCR and posi i e was iden i ied i PCR we e posi i e a wo o h ee imes. Collec ion o backg ound in o ma ion and labo a o y es s The da a on pa icipan s’socioeconomic backg ound we e col- lec ed h ough pe sonal in e iews du ing en olmen . A en ol- men , he pa icipan s’blood haemoglobin concen a ion was also measu ed om a enous sample using cu e es and a eade (HemoCue AB, Angelholm, Sweden). Mala ia was diagnosed mic oscopically om Giemsa s ained hick and hin blood ilms. Each smea was ead by wo independen mic oscopis s and disco dan esul s we e e- ead by a hi d mic oscopis . An h opome ic measu emen s An h opome ic measu emen s we e ca ied ou by h ee ained esea ch assis an s du ing he en olmen isi . Unclo hed in an s we e weighed using an elec onic in an weighing scale (SECA 735; Chasmo s L d, London, England) and weigh s we e eco ded o he nea es 10 g. The leng h was measu ed o he nea es 1 mm using a high-quali y leng h boa d (Kiddime e; Ra en Equipmen L d, Essex, England). An h opome ic indices (weigh - o -age Z sco e = WAZ, leng h- o -age Zsco e = LAZ and weigh - o -leng h Zsco e = WLZ) we e calcula ed using WHO Child G ow h S anda ds (2010 STATA ig owup package) [24]. Age o all pa i- cipan s was e i ied be o e en olmen objec i ely by c osschecking documen ed bi h da es in heal h passpo s and unde - i e ca ds. S a is ical analysis P opo ions, means and s anda d de ia ions (S.D.) we e compa ed be ween g oups by using S uden ’s es o con inuous a iables and Fishe ’s exac es o p opo ions. F equencies we e used o de e mine he p e alence o i uses and pa asi es. Bi a ia e ana- lysis was used o de e mine he associa ion be ween po en ial p e- dic o s including age, sex, g ow h indexes, age o he main gua dian, numbe o pe sons and child en <5 yea s o age in household, pa en s’educa ion le el, socioeconomic s a us, num- be o animals in he house, anaemia, mala ia, sou ce o d inking 2 Y.-M. FAN e al. h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. wa e , household sani a ion (la ine), si e (Lungwena o Malindi), seasonali y (d y o ainy season when s ool samples we e col- lec ed) and p esence o i uses and pa asi es. Those p edic o s ha we e ound o be a he P< 0.1 le el we e included in he mul i a ia e logis ic eg ession model. Age, sex, socioeconomic s a us and numbe o animals in household we e o ced in o he model since hey ha e been ound o be signi ican in he li - e a u e and a e po en ial con ounde s [25–27]. Odds a ios wi h 95% CI we e used o measu e he associa ion be ween p edic o s and p esence o i uses and pa asi es a s a is ical signi icance o P< 0.05. S a is ical analyses we e pe o med using S a a 13.1 (S a a Co p, College S a ion, TX, USA). Resul s Cha ac e is ics o s udy subjec s O he 840 LCNI-5 ial pa icipan s, we had 506 s o ed s ool samples om 6-mon h in an s. Among hem, 37 we e ound o ha e dia hoea a sample collec ion day and we e excluded om his s udy. The e we e 11 in an s who had dia hoeal du ing he pas 2 days when he samples we e collec ed. Howe e , hese pa - icipan s we e included in he s udy subjec s. S ool samples o 354 and 468 child en we e a ailable a age o 12 and 18 mon hs, espec i ely (Fig. 2). A 6 mon hs o age, he mean LAZ, WAZ and WLZ o hese pa icipan s we e −1.65, −0.79 and +0.45, espec i ely. App oxima ely 13% o hem had mala ia pa asi - aemia in hei blood. In an s who pa icipa ed in he main in e en ion ial bu we e no included in his sub-s udy we e on a e age simila o he included ones, excep ha hey we e sligh ly olde and less o en en olled om Lungwena heal h cen e (Table 1). P e alence o i uses and pa asi es in s ool samples A o al o 320 (68.2%), 284 (80.2%) and 380 (81.2%) o he s ool samples con ained en e o i al RNA a 6, 12 and 18 mon hs, espec - i ely. Rhino i us was de ec ed in 130 (27.7%), 65 (18.4%) and 146 (31.2%) samples; no o i us in 110 (23.5%), 79 (22.3%) and 74 (15.8%); pa echo i us in 106 (22.6%), 60 (17%) and 77 (16.5%); and o a i us in 15 (3.2%), 4 (1.1%) and 3 (0.6%) samples om 6, 12 and 18 mon hs, espec i ely (Table 2). A o al o 45 (9.6%), 83 (23.5%) and 123 (26.3%) samples we e posi i e o Gia dia lamblia and 30 (6.4%), 28 (7.9%) and 9 (1.9%) o C yp ospo idium (spp.) om 6, 12 and 18 mon hs, espec i ely (Table 2). A leas one i us o pa asi e was de ec ed in 414 (88.3%), 317 (89.5%) and 418 (89.3%) s ool samples a 6, 12 and 18 mon hs. Two o mo e mic obes we e de ec ed in 234 (49.9%), 188 (53.1%) and 262 (56%) and h ee o mo e mic obes in 86 (18.3%), 72 (20.7%) and 105 (22.4%) o 6, 12 and 18 mon hs samples. We also used C alues o classi y in ec ions as low (C ⩾36), median (30 ⩽C ⩽36) and high (C < 30) le els. The p e alence o mic obes is highes in high in ec ion le els in mos o he i uses excep hino i us in which he low in ec ion le el is he highes (da a no shown). Fig. 1. Map o s udy si es (Lungwena and Malindi). Sou ce: Da a laye o A ica map downloaded om h p://www. hema icmapping.o g, 2015 a ailable unde a C ea i e Commons A ibu ion-Sha e Alike License 3.0 (bo de s may no be comple ely accu a e). All o he da a laye s downloaded om Malawi Spa ial Da a Po al, 2015 (h p://www.masdap.mw). Epidemiology and In ec ion 3 h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. P edic o s o i al and pa asi ic in ec ions a 6-mon in an s In bi a ia e analyses, en e o i us posi i i y in he s ool samples was signi ican ly associa ed wi h sani a ion (no household la ine use), si e (li ing in Lungwena), lowe le el o mo he ’s and a he ’s educa- ion and ainy season (Table 3). In mul i a ia e analysis, seasonali y and a he ’s educa ion le el emained signi ican ly associa ed wi h en e o i us posi i i y (Table 3). In an s whose a he had a highe le el o educa ion we e less likely en e o i us posi i e han hose whose a he had a lowe le el o educa ion (OR 0.94, 95% CI 0.89–0.99, P= 0.031). In an s whose sample was collec ed du ing he d y season (May–Oc obe ) we e less likely o be en e o i us posi- i e han hose whose sample was collec ed du ing he ainy season (No embe –Ap il) (OR 0.65, 95% CI 0.43–0.99, P=0.045) (Table 3). Agewas associa ed wi h hino i us in ec ions in mul i a i- a e analysis. Olde in an s we e less likely hino i us posi i e han hose young in an s (OR 0.43, 95% CI 0.19–0.99,P= 0.046) (Table 3). The esul s o no o i us, pa echo i us and o a i us a e shown in Table 4. In bi a ia e analysis, he numbe o pe sons in he household, mo he ’s educa ion le el, a he ’s educa ion le el, anaemia and ainy season we e signi ican ly associa ed wi h no o i us p esence. Anaemia was associa ed wi h pa echo i us in ec ions and lowe le el o sani a ion (household la ine use) was associa ed wi h o a- i usin ec ions (Table 4). In mul i a ia e analysis, numbe o pe sons in he household and seasonali y emained signi ican ly associa ed wi h no o i us p esence; and sani a ion wi h o a i us p esence. The mo e pe son li ing in he house, he mo e likely in an s we e o ha e no o i us in ec ion (OR 1.16, 95% CI 1.02–1.31, P=0.024). In an s whose sample was collec ed du ing he d y season (May– Oc obe ) we e less likely no o i us posi i e han hose whose sample was collec ed du ing he ainy season (No embe –Ap il) (OR 0.53, 95% CI 0.32–0.86, P= 0.01). In an s whose home did no ha e la ine had ou imes highe odds o o a i us being de ec ed in hei s ool han hose whose home had la ine (OR 4.11,95% CI 1.13–14.88,P= 0.032) (Table 4). Fo Gia dia lamblia in ec ion, he s udy si e and mo he ’s edu- ca ion le el we e associa ed wi h i in bi a ia e analysis (P= 0.015 and P= 0.012). In mul i a ia e analysis, bo h associa ions we e disappea ed (P= 0.097 and P= 0.082) (Table 5). C yp ospo idium (spp.) in ec ions we e signi ican ly associa ed wi h seasonali y and WLZ in bi a ia e analysis. In mul i a ia e ana- lysis, hey emained independen ly associa ed wi h C yp ospo id- ium in ec ions oge he wi h gende (Table 5). In an s who had highe WLZ we e less likely in ec ed wi h C yp ospo idium han hose wi h lowe WLZ (OR 0.58, 95% CI 0.39–0.87, P= 0.008). In an s whose samples we e collec ed du ing he d y season (May–Oc obe ) we e less likely o be in ec ed wi h C yp ospo idium han hose whose samples we e collec ed du ing he ainy season (No embe –Ap il) (OR 0.3, 95% CI 0.11–0.81, P= 0.018). Gi ls we e mo e likely in ec ed wi h C yp ospo idium han boys (OR 2.86, 95% CI 1.19–6.88, P= 0.019). Discussion Ou s udy in es iga ed he p e alence o selec ed common i uses and pa asi es in a popula ion o asymp oma ic 6, 12 and 18- Fig. 2. Flow diag am o pa icipan p og ess h oughou he s udy. CSB, co n-soy blend; LAZ, leng h- o -age Z-sco es; milk-LNS, lipid-based nu ien supplemen wi h milk p o ein base; soy-LNS, lipid-based nu ien supplemen wi h soy p o ein base; WFL, weigh - o -leng h. 4 Y.-M. FAN e al. h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. mon h-old Malawian child en. The s udy also assessed he en i - onmen al p edic o s o he p esence o i uses and pa asi es among 6-mon h in an s. We ound a high p e alence o i al and pa asi ic in ec ions in hese child en. Se e al p edic o s we e ound o be associa ed wi h he in ec ions including season- ali y o sample collec ion, sani a ion condi ions and a he ’s edu- ca ion le el. In his s udy, we used eal- ime PCR o de ec i uses and pa asi es. Due o he sensi i i y o hese assays we could de ec also low-g ade in ec ions which may be common in asymp om- a ic in an s and child en. Compa ed wi h adi ional me hods such as cell cul u e, an igen de ec ion o mic oscopic examina ion o i uses o pa asi es, ou esul s gi e mo e ep esen a i e pic u e o he epidemiology o hese in ec ions including asymp oma ic ones. The p e alence o i uses and pa asi es in ou s udy we e much highe han hose s udies ca ied ou in high-income coun- ies, and mo e compa able wi h s udies in low-income coun ies. The en e o i us p e alence in ou s udy was much highe han hose epo ed in No wegian [28] and Finnish in an s [13,29], bu mo e compa able wi h Mongolia in an s [30]. The same end was o he p e alence o hino i us in ou s udy s. Finnish in an s [21,29]; pa echo i us s. Finnish in an s [13, 31] and No wegian in an s [32]; no o i us s. Ge man [33], Ko ean [34], Came oonian [12] and Sou h A ican child en [35]. The p e alence o Gia dia lamblia in ou s udy was also highe han epo ed in Bangladeshi heal hy in an s [36], bu qui e compa able wi h Ugandan asymp oma ic in an s [37]. C yp ospo idium (spp.) p e alence was also highe han p e i- ously epo ed om Spanish [38] and F ench child en [14], and Chinese [15] and Bangladeshi in an s [36]. Ou esul s we e also compa able wi h hose in MAL-ED and GEMS non- dia hoeal samples in no o i us [16,18] and Gia dia in ec ions [17], bu wi h a lowe a e o o a i us and C yp ospo idium in ec- ions in ou s udy [18]. The disc epancy be ween ou indings and hose o o he s ud- ies can o some ex en be explained by he use o di e en me h- ods o de ec ing he in ec ions. In addi ion, o he ac o s such as di e ences in s udy popula ions, pe sonal and communi y hygienic s anda ds, sani a ion, clima e, home c owding, wa e and ood supplies, socioeconomic condi ion, seasonal a ia ions, p oximi y o bo h domes ic and wild animals and a high p e a- lence o o he diseases and condi ions such as HIV in ec ion and malnu i ion may ha e in luenced he indings in di e en s udies. Only a ew s udies ha e in es iga ed he p edic o s o i al and pa asi ic in ec ions in heal hy in an s li ing in u al low-income se ings. Ou esul s a e, howe e , in ag eemen wi h p e ious s udies showing seasonali y o en e o i us and C yp ospo idium in ec ions [39–41]. The e is also some p e ious e idence om Acc a ha he use o non- lush oile s is associa ed wi h a highe en e o i al isola ion a e among heal hy in an s [39]. The absence o la ines has been associa ed wi h dia hoea incidence also in Vie nam, whe e o a i us was he mos common causa i e agen [42]. These indings a e consis en wi h he ecal-o al ans- mission ou e o hese mic obes and sugges ha imp o emen s in sani a y condi ions migh dec ease he p e alence o en e ic i al in ec ions in u al Malawi. Ou s udy shows he high asymp oma ic in ec ions in Malawian child en. These child en ca ying he pa hogens did Table 1. Cha ac e is ics o pa icipan s S udy pa icipan s (N= 469) Subjec s wi hou s ool samples o wi h dia hoea (N= 371) P alue P opo ion o boys 47.3% 53.1% 0.110 Mean (S.D.) in an age, mon hs 5.98 (0.25) 6.09 (0.22) <0.001 Mean (S.D.) weigh - o -age z-sco e (WAZ) −0.79 (1.10) −0.79 (1.13) 0.499 Mean (S.D.) leng h- o -age z-sco e (LAZ) −1.65 (1.00) −1.66 (1.03) 0.4403 Mean (S.D.) weigh - o -leng h z-sco e (WLZ) 0.45 (1.02) 0.45 (1.08) 0.473 Mean (S.D.) age o he main gua dian, yea s 28 (9) 28 (10) 0.342 Mean (S.D.) numbe o pe sons in household 4.8 (1.8) 4.9 (1.8) 0.196 Mean (S.D.) numbe o child en <5 yea s o age in household 1.6 (0.7) 1.6 (0.6) 0.145 Mean (S.D.) mo he s’educa ion, comple ed yea s 3.5 (3.4) 3.6 (3.2) 0.328 Mean (S.D.) a he s’educa ion, comple ed yea s 4.1 (3.8) 4.3 (4.0) 0.269 Mean (S.D.) numbe o economic suppo pe son in he amily 1.5 (1.1) 1.7 (1.3) 0.076 Mean (S.D.) numbe o animals in he house 5.9 (10.6) 5.2 (8.0) 0.166 P opo ion wi h mode a e anaemia (Hb <80 g/l) 15.0% 14.9% 1.00 P opo ion wi h posi i e mala ia mic oscopy 13.0% 14.6% 0.539 P opo ion wi h unsa e sou ce o d inking wa e a 10.2% 6.2% 0.056 P opo ion wi h household la ine 92.8% 93.8% 0.673 P opo ion en olled om Lungwena si e 62.9% 48.5% <0.001 P opo ion o sample collec ion a ainy season, No embe –Ap il 58.4% 53.6% 0.183 S.D., s anda d de ia ion. a Sou ce o d inking wa e om unp o ec ed well, lake o pond. P- alue ob ained om S uden ’s es (con inuous a iables) o Fishe ’s exac es (p opo ions). Epidemiology and In ec ion 5 h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. no ha e clinical symp oms and looked ‘heal hy’ om hei ca e ake s’ iews when he s ool samples we e collec ed. I is pos- sible ha he high p e alence o he asymp oma ic in ec ions in hese child en is due o he equen exposu e o he pa hogens which a e common in low-income se ings. In ega ds o C yp ospo idium and Gia dia lamblia, some species does no in ec human and possibly play a ole in he high p e alence o pa asi ic asymp oma ic in ec ions in ou s udy. To educe he Table 3. Bi a ia e and mul i a ia e analysis o po en ial p edic o s o en e o i us and hino i us among he pa icipan s En e o i us bi a ia e En e o i us mul i a ia e Rhino i us bi a ia e Rhino i us mul i a ia e Va iable OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue Age 0.50 (0.23–1.10) 0.084 0.64 (0.27–1.53) 0.32 0.46 (0.20–1.03) 0.06 0.43 (0.19–0.99) 0.046 Gende (gi ls) 1.15 (0.78–1.69) 0.491 1.19 (0.79–1.79) 0.418 1.11 (0.74–1.67) 0.6 1.16 (0.77–1.76) 0.485 Numbe o economic suppo pe son in he amily 1.02 (0.84–1.23) 0.849 1.09 (0.90–1.33) 0.374 1.05 (0.87–1.27) 0.612 1.06 (0.87–1.28) 0.573 Sani a ion (no household la ine) 2.72 (1.03–7.20) 0.044 2.40 (0.87–6.59) 0.089 0.68 (0.29–1.62) 0.385 Si e (Lungwena) 1.56 (1.05–2.32) 0.028 1.18 (0.75–1.87) 0.467 1.16 (0.76–1.77) 0.49 Numbe o child en < 5 yea s o age in household 1.28 (0.96–1.71) 0.096 1.19 (0.88–1.59) 0.258 1.12 (0.85–1.47) 0.435 High ma e nal educa ion le el 0.91 (0.86–0.97) 0.002 0.96 (0.89–1.02) 0.208 0.99 (0.93–1.05) 0.665 High pa en al educa ion le el 0.92 (0.87–0.97) 0.001 0.94 (0.89–0.99) 0.031 0.99 (0.94–1.04) 0.664 Season (May–Oc obe ) 0.64 (0.43–0.95) 0.027 0.65 (0.43–0.99) 0.045 1.04 (0.69–1.57) 0.843 Numbe o animals in house 0.89 (0.75–1.07) 0.209 1.00 (0.98–1.02) 0.791 0.99 (0.97–1.01) 0.404 0.99 (0.97–1.01) 0.403 CI, con idence in e al; OR, odds a io. Bi a ia e analysis also included WAZ, LAZ, WLZ, numbe o pe sons in household, anaemia, mala ia, sou ce o d inking wa e and age o main gua dian (N= 455–469). Table 2. P e alence o i uses and pa asi es in 6, 12 and 18-mon h-old Malawian child en Vi us/Pa asi e Age NNo posi i e % o posi i e 95% CI En e o i us 6-mon h 469 320 68.2 64.0–72.5 12-mon h 354 284 80.2 76.1–84.4 18-mon h 468 380 81.2 77.6–84.7 Rhino i us 6-mon h 469 130 27.7 23.7–31.8 12-mon h 354 65 18.4 14.3–22.4 18-mon h 468 146 31.2 27.0–35.4 No o i us 6-mon h 469 110 23.5 19.6–27.3 12-mon h 354 79 22.3 18.0–26.7 18-mon h 468 74 15.8 12.5–19.1 Pa echo i us 6-mon h 469 106 22.6 18.8–26.4 12-mon h 354 60 17.0 13.0–20.9 18-mon h 468 77 16.5 13.1–19.8 Ro a i us 6-mon h 469 15 3.2 1.6–4.8 12-mon h 354 4 1.1 0.02–2.2 18-mon h 468 3 0.6 −0.08 o 1.4 Gia dia lamblia 6-mon h 469 45 9.6 6.9–12.3 12-mon h 354 83 23.5 19.0–27.9 18-mon h 468 123 26.3 22.3–30.3 C yp ospo idium 6-mon h 469 30 6.4 4.2–8.6 12-mon h 354 28 7.9 5.1–10.7 18-mon h 468 9 1.9 0.7–3.2 CI, con idence in e al. 6 Y.-M. FAN e al. h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. Table 4. Bi a ia e and mul i a ia e analysis o po en ial p edic o s o no o i us, pa echo i us and o a i us among he pa icipan s No o i us bi a ia e No o i us mul i a ia e Pa echo i us bi a ia e Pa echo i us mul i a ia e Ro a i us bi a ia e Ro a i us mul i a ia e Va iable OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue Age 1.29 (0.55–3.02) 0.56 1.40 (0.58–3.34) 0.455 1.38 (0.58–3.28) 0.463 1.44 (0.61–3.44) 0.407 0.57 (0.07–4.38) 0.588 0.82 (0.10–6.97) 0.859 Gende (gi ls) 1.16 (0.75–1.78) 0.503 1.08 (0.68–1.71) 0.753 1.06 (0.69–1.63) 0.795 1.03 (0.65–1.62) 0.902 1.03 (0.37–2.88) 0.958 1.28 (0.43–3.79) 0.652 Numbe o economic suppo pe son in he amily 0.98 (0.80–1.21) 0.87 1.07 (0.86–1.34) 0.552 1.05 (0.85–1.28) 0.669 1.10 (0.89–1.35) 0.384 0.93 (0.55–1.57) 0.79 1.00 (0.59–1.71) 0.992 Sani a ion (no household la ine) 0.88 (0.37–2.10) 0.778 1.31 (0.59–2.92) 0.503 5.23 (1.57–17.44) 0.007 4.11 (1.13–14.88) 0.032 High ma e nal educa ion le el 0.92 (0.86–0.99) 0.017 0.95 (0.87–1.03) 0.183 1.02 (0.95–1.09) 0.579 0.88 (0.73–1.05) 0.16 High pa en al educa ion le el 0.93 (0.88–0.99) 0.022 0.94 (0.88–1.01) 0.094 1.01 (0.96–1.07) 0.611 0.85 (0.72–1.01) 0.059 0.88 (0.74–1.04) 0.128 Season (May-Oc obe ) 0.55 (0.35–0.87) 0.01 0.53 (0.32–0.86) 0.01 0.66 (0.42–1.04) 0.072 0.71 (0.44–1.13) 0.149 1.63 (0.58–4.58) 0.352 Numbe o animals in house 0.98 (0.96–1.01) 0.199 0.98 (0.95–1.01) 0.169 0.99 (0.97–1.02) 0.512 0.99 (0.97–1.02) 0.461 0.95 (0.86–1.05) 0.307 0.96 (0.87–1.06) 0.44 Numbe o pe sons in household 1.13 (1.01–1.27) 0.032 1.16 (1.02–1.31) 0.024 1.06 (0.94–1.19) 0.322 0.92 (0.68–1.25) 0.6 Anaemia 0.45 (0.22–0.95) 0.035 0.45 (0.20–1.01) 0.052 0.47 (0.23–0.99) 0.046 0.52 (0.24–1.10) 0.086 1.57 (0.43–5.76) 0.5 CI, con idence in e al; OR, odds a io. Bi a ia e analysis also included WAZ, LAZ, WLZ, si e, numbe o child en <5 yea s o age in household, age o main gua dian and sou ce o d inking wa e (N= 455–469). Epidemiology and In ec ion 7 h ps://doi.o g/10.1017/S0950268819000025 Downloaded om h ps://www.camb idge.o g/co e. Uni e si y o Tampe e, on 17 Ap 2019 a 09:09:07, subjec o he Camb idge Co e e ms o use, a ailable a h ps://www.camb idge.o g/co e/ e ms. in ec ion a e based on ou indings, hygiene, be e sani a ion especially in he ainy season a e impo an . Taken oge he , ou indings e eal a high p e alence o i uses and pa asi es in asymp oma ic Malawian child en. The high p e alence o in ec ion could a ec immune esponse and p oduc ion o p o-in lamma o y cy okines, nu i ional s a us and he de elopmen o EED. Ch onic in ec ions may be asymp- oma ic in hese child en. Howe e , he cumula i e e ec o hese in ec ions could be subs an ial. Fu u e s udies a e needed o in es- iga e i such in ec ions and speci ic species o pa hogens a e asso- cia ed wi h he de elopmen o EED and e en ually lead o g ow h ailu e among in an s and child en in low-income se ings. Supplemen a y ma e ial. The supplemen a y ma e ial o his a icle can be ound a h ps://doi.o g/10.1017/S0950268819000025. Au ho ORCIDs. Y.-M. Fan, 0000-0002-3241-6225 Acknowledgemen s. We hank he s udy pa icipan s and he s a membe s o Lungwena Heal h Cen e in Mangochi Dis ic o hei suppo and help in he s udy and Emily McKinney, Ma ie Hélou, Ma ia O askainen, E eliina Jalonen and Basho Poelman o hei skil ul echnical assis ance. The LCNI-5 clinical ial was unded by he Academy o Finland (g an s 109796 and 127025), wi h addi ional unding om he Founda ion o Paedia ic Resea ch in Finland, he compe i i e esea ch Funding o he Tampe e Uni e si y Hospi al (g an s 9H003, 9J006 and 9R005), he US Agency o In e na ional De elopmen unde e ms o coope a i e ag eemen CHN-A- 00-08-00001-00, h ough he Food and Nu i ion Technical Assis ance-2 P ojec managed by he Academy o Educa ional De elopmen (AED) and he Nu ise Inc. Con lic o in e es . None. Re e ences 1. Black RE e al.(2013) Ma e nal and child unde nu i ion and o e weigh in low-income and middle-income coun ies. Lance 382, 427–451. 2. Vic o a CG e al.(2008) Ma e nal and child unde nu i ion: conse- quences o adul heal h and human capi al. Lance 371, 340–357. 3. Keusch GT e al.(2014) En i onmen al en e ic dys unc ion: pa hogenesis, diagnosis, and clinical consequences. 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Jou nal o Medical Vi ology 85, 1678– 1684. Table 5. Bi a ia e and mul i a ia e analysis o po en ial p edic o s o Gia dia lamblia and C yp ospo idium among he pa icipan s Gia dia bi a ia e Gia dia mul i a ia e C yp ospo idium bi a ia e C yp ospo idium mul i a ia e Va iable OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue OR (95% CI) P alue Age 0.91 (0.27–3.10) 0.882 1.19 (0.35–4.03) 0.781 0.82 (0.19–3.59) 0.795 1.78 (0.43–7.37) 0.424 Gende (gi ls) 0.93 (0.50–1.73) 0.826 1.01 (0.53–1.89) 0.987 2.20 (0.99–4.91) 0.054 2.86 (1.19–6.88) 0.019 Numbe o economic suppo pe son in he amily 0.78 (0.55–1.11) 0.172 0.87 (0.60–1.25) 0.439 1.03 (0.73–1.45) 0.877 1.16 (0.80–1.69) 0.433 Sani a ion (no household la ine) 1.30 (0.44–3.89) 0.636 0.43 (0.06–3.26) 0.414 Si e (Lungwena) 2.55 (1.20–5.43) 0.015 1.97 (0.88–4.39) 0.097 2.48 (0.99–6.19) 0.052 2.59 (0.97–6.89) 0.057 Numbe o child en <5 yea s o age in household 1.22 (0.83–1.80) 0.301 0.88 (0.52–1.51) 0.645 High ma e nal educa ion le el 0.87 (0.78–0.97) 0.012 0.90 (0.81–1.01) 0.082 0.95 (0.84–1.07) 0.359 High pa en al educa ion le el 0.94 (0.87–1.03) 0.185 0.97 (0.88–1.08) 0.619 Season (May–Oc obe ) 0.61 (0.31–1.17) 0.137 0.33 (0.13–0.83) 0.018 0.30 (0.11–0.81) 0.018 Numbe o animals in house 0.99 (0.95–1.02) 0.489 0.99 (0.95–1.03) 0.644 0.99 (0.94–1.03) 0.537 0.97 (0.91–1.04) 0.393 WLZ 0.96 (0.71–1.30) 0.801 0.68 (0.47–0.99) 0.043 0.58 (0.39–0.87) 0.008 Age o main gua dian 1.01 (0.97–1.04) 0.665 1.03 (1.00–1.07) 0.08 1.04 (0.99–1.09) 0.051 CI, con idence in e al; OR, odds a io. 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