Breast-Feeding Protects Infantile Diarrhea Caused by Intestinal Protozoan Infections

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1 ISSN (Print) ISSN (Online) ORIGINAL ARTICLE Koren J Prsitol Vol. 51, No. 5: , October Brest-Feeding Protects Infntile Dirrhe Cused by Intestinl Protozon Infections Ekhls Hmed Abdel-Hfeez 1, *, Usm Slh Bell 1, Mnl Zki Mohmed Abdelltif 1, Koji Noi 2 nd Kzumi Norose 3 1 Deprtment of Prsitology, Fculty of Medicine, Mini University, Mini, 61519, Egypt; 2 Medicl Corportion Kohitsujiki, Chib, , Jpn; 3 Deprtment of Infection nd Host Defense, Grdute School of Medicine, Chib University, Chib, , Jpn Abstrct: This study investigted the effect of brest-feeding in protection ginst protozon infection in infnts with persistent dirrhe. Infnts were clssified into 2 groups; 161 brest-fed infnts nd the sme number of non-brest-fed infnts. Microscopic exmintions of stool were done for detection of prsites nd mesuring the intensity of infection. Moreover, serum levels of IgE nd TNF-α were mesured by ELISA. Cryptosporidium spp., Entmoeb histolytic/entmoeb dispr, Girdi lmbli, nd Blstocystis sp. were demonstrted in infnts with persistent dirrhe. The percentge of protozon infections ws significntly lower in brest-fed infnts thn tht in the non-brest-fed infnts. The levels of IgE nd TNF-α were significntly lower in the brest-fed group thn in the non-brest-fed group. There were significnt positive ssocitions between the serum levels of IgE nd TNF-α nd the intensity of prsite infection in the brest-fed group. It is suggested tht brest-feeding hs n ttenuting effect on the rte nd intensity of prsite infection. Key words: Entmoeb histolytic, Entmoeb dispr, Girdi lmbli, Cryptosporidium, Blstocystis, brest-feeding, brest milk, dirrhe, intestinl protozo, IgE, TNF-α INTRODUCTION Protozon prsites such s Cryptosporidium spp., Entmoeb histolytic, nd Girdi lmbli [1] cuse dirrhe which still remins leding cuse of morbidity nd mortlity mong infnts in low- nd middle-income countries [2]. As infections with prsites, prticulrly G. lmbli nd Cryptosporidium spp., re the most likely cuses of persistent dirrhe (>14 dys) [3], peditricins hd strong suspicion from the symptoms nd clinicl courses of these infnts tht the cuses of persistent dirrhe should be intestinl protozon infections (IPI), i.e., infections cused by pthogenic protozo in the intestine [4]. Brest-feeding provides significnt protection ginst mny diseses including dirrhe in infncy [5]. This is ttributble to complex of cquired nd innte fctors unique to humn milk including immunoglobulins [6,7], oligoscchrides, glycoconjugtes, lctoferrin, ntimicrobil compounds [8], leukocytes, cytokines, nd other gents [9,10]. Brest-fed infnts Received 2 My 2013, revised 20 August 2013, ccepted 21 August * Corresponding uthor (ekhlsh@yhoo.com) 2013, Koren Society for Prsitology nd Tropicl Medicine This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. hve lower prevlence of gstrointestinl infections including IPI thn formul-fed infnts [9,10]. However, there re little conflicting opinions concerning brest-milk nd nti-infective functions [11,12]. Detiled investigtions re still limited. IgE is considered to ply centrl role in protective immunity ginst prsites, not only helminths [13] but lso some protozo such s G. intestinlis (synonymous with G. lmbli nd G. duodenlis) [14]. TNF-α is n importnt inflmmtory cytokine in immune regultion nd resistnce to vrious microbes including protozo [15]. This cytokine is known to be of significnt importnce in the pthogenesis of inflmmtory bowel diseses, nd nti- TNF gents hve proven to be effective in the tretment of chronic ctive inflmmtory bowel diseses nd fistulizing disese [16]. Thus, the present study ws designed to investigte the brest-feeding in protection ginst IPI by compring brestfed with non-brest-fed infnts with persistent dirrhe. MATERIALS AND METHODS Subjects nd methods This study ws crried out with 322 infnts (146 boys nd 176 girls, 2-6 months old) who presented with persistent dir- 519

2 520 Koren J Prsitol Vol. 51, No. 5: , October 2013 rhe (minimum of 3 loose stools/dy for more thn 14 dys) in spite of n nti-bcteril drug, nifuroxzide. They hd been enrolled from inptient wrds nd outptient clinics of the Peditric Deprtment of University Hospitl, Mini District, Egypt, between Jnury 2011 nd Mrch These infnt cses were divided into 2 equl groups of 161 ech. The first group comprised of 161 prtilly brest-fed infnts (79 boys nd 82 girls, men±sd=4.5±1.1 months old) nd the second n equl number of non-brest-fed infnts (some were hving formul lone, nd some bovine milk, 67 boys nd 94 girls, 4.4±1.2 months old). There ws no difference in sex between these 2 groups. The smple chrcteristics (ge, prent socio-economic levels, nd residency) of these 2 groups were similr. The brest-fed infnts hd been brest-fed since birth, while non-brest-fed infnts hd received formul since 1 or 2 weeks fter birth. Formul ws dissolved in tp wter, which hd been boiled nd cooled down. Ethicl considertions Verbl consent ws obtined from the prents of these infnts. All procedures were conducted ccording to the ethicl stndrds pproved by the Institutionl Humn Ethics Committee, Fculty of Medicine, Mini University, Egypt. Stool exmintions Stool smples were collected in clen plstic continer once per infnt but severl stool smples were collected on lternte dys to detect prsites becuse cysts of G. lmbli re excreted intermittently. Fecl specimens were trnsported to the lbortory of Prsitology Deprtment, Fculty of Medicine, Mini University, Egypt to be exmined by different techniques for prsitic infections within 1-3 hr fter collection. For microscopic exmintions, direct wet smer methods, nmely sline wet mount nd iodine wet mount, were pplied. The technique of formol-ether sedimenttion ws used to concentrte the cysts or oocysts of the protozo. A sme mount of stools (50 mg) from ech of ll infnts ws mixed with 10 ml of fixtion buffer (sodium-cette cetic cid formlin, SAF) nd incubted for 1 hr for fixtion nd inctivtion. The suspension ws pssed through 4 lyers of cotton netting nd centrifuged t 2,000 g for 5 min. Two smers were dried in ir, fixed with methnol, nd then exmined using 2 different stins: cid fst stin (modified Ziehl-Neelsen stin) for identifiction of Cryptosporidium spp. nd Giems stin for identifiction of Blstocystis sp. The intensity of infection ws scored s follows by 1,000 mgnifiction: 1, very low infection (1 prsite per field); 2, mild infection (2 prsites per field); 3, moderte infection (3 prsites per field); 4, hevy infection (4 or more prsites per field) [17]. Blood smple collection Blood smples were collected from rm vein nd ser were stored t -20 C until use. The serum levels of IgE nd TNF-α were mesured using commercilly vilble ELISA kits (MYM Lbortory nd Medicl Supply, Inc., Sn Diego, Cliforni, USA, nd Anogen, Ontrio, Cnd, respectively) ccording to the mnufcturers' guidelines. Sttisticl nlysis Dt were coded nd verified prior to dt entry. No dt were double-entered. As the dt were distributed s norml, results of the studies were reported s men± SD. The Sttisticl Pckge of SPSS version 16 for Windows ws used for dt entry nd nlysis. Descriptive sttistics were clculted. The χ 2 -test ws used to compre proportions of prsite detection between groups nd of sex difference between groups. The Student s t-test ws used to compre mens between groups. Regression nlysis ws used for estimting the reltionships between serum levels of IgE nd TNF-α nd the intensity of the infection. P < 0.05 ws considered to be sttisticlly significnt. RESULTS Prsite detection nd infection intensity mong infnts with persistent dirrhe Protozo were detected in 62 brest-fed infnts (38.5%), significntly lower incidence thn tht in non-brest-fed infnts, mong which 121 (75.2%) were infected (Fig. 1). Cryptosporidium spp. ws the most common protozon detected in both groups, followed by E. histolytic/e. dispr, G. lmbli, nd Blstocystis sp. Mixed prsitic infections were observed only in the non-brest-fed group. The intensity of single prsitic infection of the brest-fed group ws significntly less thn tht of the non-brest-fed group (Tble 1). Serum levels of IgE nd TNF-α in infnts with persistent dirrhe Serum levels of IgE nd TNF-α were summrized in Tble 2. In the brest-fed group, the serum levels of IgE nd TNF-α of

3 Abdel-Hfeez et l.: Brest-feeding protects dirrhe cused by protozon infections 521 Tble 1. Prsite intensity of infected infnts with persistent dirrhe ccording to feeding stte Brestfed Nonbrest-fed Totl Prsite intensity Single prsite infection 2.9± ± ± 1.0 Mixed prsite infections - 2.7± ± 1.0 Totl 2.9± ± ± 1.0 P < 0.05 between brest-fed vs. non-brest-fed. Tble 2. The levels of serum IgE nd TNF-α in infnts with persistent dirrhe Protozo Brest-fed (n=161) IgE (IU/ml) TNF-α (pg/ml) Non-brest-fed (n=161) IgE (IU/ml) TNF-α (pg/ml) Positive 514.8±95.2,b 6.2±0.7,b 645.9±67.8 b 7.7±1.2 b (n=183) Boys 487.8± ±0.8 c 653.1± ±1.1 (n=72) Girls 534.3± ± ± ±1.2 (n=111) Negtive (n=139) 118.1± ± ± ±0.3 P < 0.01 between brest-fed nd non-brest-fed group. b P < 0.01 between positive nd negtive group. c P < 0.05 between boys nd girls. Fig. 1. Intestinl protozo isolted from feces of 322 infnts with persistent dirrhe ccording to feeding stte. The detiles of the mixed prsite infection from non-brest-fed group were s follows: Cryptosporidium spp. nd E. histolytic/dispr; 4 (3.3%), Cryptosporidium spp. nd G. lmbli; 4 (3.3%), nd Cryptosporidium spp. nd Blstocystis sp.; 6 (5.0%). P < 0.05 between brest-fed vs. non-brest-fed. prsite positive infnts were significntly elevted (514.8 ± 95.2 IU/ml nd 6.2±0.7 pg/ml, respectively) compred with the levels in prsite negtive infnts (118.1±15.9 IU/ml nd 3.5±0.5, respectively) (P <0.01). In non-brest-fed group, the serum levels of IgE nd TNF-α of prsite positive infnts were lso significntly elevted (645.9±67.8 IU/ml nd 7.7±1.2 pg/ml, respectively) thn those of prsite negtive infnts (121.1±9.6 IU/ml nd 3.7±0.3 pg/ml, respectively) (P <0.01). In ddition, the serum levels of IgE nd TNF-α of prsite-infected infnts were significntly lower in the brest-fed group (514.8±95.2 IU/ml nd 6.2±0.7 pg/ml, respectively) thn those in the non-brest-fed group (645.9 ±67.8 IU/ml nd 7.7±1.2 pg/ml, respectively) (P <0.01). The serum levels of IgE nd TNF-α of infnts infected with ech prsite were significntly lower in the brest-fed group thn in the non-brestfed group (P <0.01) (dt not shown). There were no significnt differences in the serum levels of IgE nd TNF-α of the prsite-non-infected infnts between the brest-fed nd nonbrest-fed groups. There were no significnt differences in the serum levels of IgE between prsite-infected boys (487.8±97.2 IU/ml nd 653.1±67.3 IU/ml, respectively) nd girls (534.3±90.0 IU/ml nd 641.7±68.2 IU/ml) of the prsite infected brest-fed nd non-brest-fed groups. However, the serum levels of TNF-α in the brest-fed group were significntly lower in prsite-infected boys (5.8±0.8 pg/ml) thn in prsite-infected girls (6.5± 0.6 pg/ml) (P <0.05). Reltion between serum levels of IgE nd TNF-α nd prsite infection intensity There ws significnt positive correltion between the infection intensity nd the serum levels of IgE (Fig. 2A) nd TNF-α (Fig. 2B) in the brest-fed group. On the other hnd, in the non-brest-fed group, there ws positive correltion be-

4 522 Koren J Prsitol Vol. 51, No. 5: , October 2013 A Serum level of IgE (IU/ml) y=64.3x R 2 = 0.4 y=25.1x R 2 = Intensity of prsite infection B Serum level of TNF-α (pg/ml) y=0.55x R 2 = 0.5 y=0.28x R 2 = Intensity of prsite infection Fig. 2. Serum levels of IgE nd TNF-α nd intensity of prsite infection. (A) IgE. (B) TNF-α. Open circles, brest-fed group; filled circles, non-brest-fed group. X, infnts infected with mixed prsites. Vlues re mens± SD. P < 0.01 between brest-fed nd non-brestfed group in sme intensity of prsite infections. tween them, lthough there were no significnt differences. The serum level of IgE tended to be higher in infnts infected with 2 prsites thn in those with single prsite in the nonbrest-fed group. With regrd to the comprison in the sme infection intensity, the levels of IgE nd TNF-α in the brestfed group were significntly lower thn those in the non-brestfed group (P <0.01) (Fig. 2). DISCUSSION Persistent dirrhe should prompt investigtion for protozon infections, minly of Girdi nd Cryptosporidium [3]. Protozon prsites, which were detected in the study, were the min cuses of dirrhe. Dirrhe ws not with n cute onset nd ws not ssocited with high fever or vomiting. Infnts, infected with protozon prsites, were treted successfully with nti-protozon drugs. This study clerly indicted tht the percentge of protozon prsite-infected infnts in the brest-fed group ws significntly less thn tht of the non-brest-fed group. The numbers of infnts infected with the sme protozon prsites in the brest-fed group were lso significntly lower thn those of the non-brest-fed group. In ddition, the intensities of prsite infections were less in the brest-fed group thn those in the non-brest-fed group. Incresed risks of persistent dirrhe re environment-relted, such s poor hygiene [18]. In this study, both mcroenvironmentl mesures (provision of sfe nd dequte wter supply, nd hygienic wste disposl) nd microenvironmentl mesures (hnd wshing, hygienic storge nd use of wter, nd voidnce of stle/cold-storge infnt foods in tropicl environments) [18] were poor in both groups. Lck of ccess to clen wter, snittion, nd hygiene re strong drivers for IPI [4]. Thus, scling-up environmentl control mesures nd sfe wter nd hygiene strtegies is importnt to prevent children from persistent dirrhe [18]. IgE ctivtes pltelets nd induces cytotoxic functions ginst prsites [19]. An elevtion in totl IgE levels ws lso reported even in helminth nd protozon infectious diseses such s

5 Abdel-Hfeez et l.: Brest-feeding protects dirrhe cused by protozon infections 523 cryptosporidiosis nd girdisis [13,14,20-22]. Excretory nd secretory proteins relesed by G. intestinlis re responsible for production of specific IgE [14]. In this study, the serum level of IgE ws significntly elevted in both prsite-infected groups compred to the non-infected infnts. The IgE levels of infnts infected with the respective prsites of the brest-fed group were significntly lower thn those of the non-brest-fed group in this study. The IgE level nd the intensity of prsite infections were relted in the brest-fed group. In ddition, the IgE level of the brest-fed group ws lower thn tht of the nonbrest-fed group with the sme intensity of prsite infections. Some fctors such s immune ctivtion by protozo might be different between the brest-fed nd non-brest-fed groups. This will be the subject of future study. The higher totl IgE levels in non-brest-fed infnts were not due to cow s milkspecific IgE; such ntibodies could not be detected in cow smilk infnt group [23]. Regrding the reltion of brest-feeding nd IgE levels in the erly childhood of helthy infnts, there re some contrdictory reports. Totl IgE level ws significntly lower in brest-fed infnts compred with formul-fed infnts, while nother report showed tht no feeding-relted differences in the IgE level in the first 6 months of life [24,25]. Protozon infections of the intestinl tissue will led to robust inflmmtory process nd production of wide rnge of cytokines including TNF-α tht is elevted in the gstrointestinl trct of some forms of inflmmtory colitis, cryptosporidiosis, E. histolytic-relted dirrhe, nd girdisis [26-30]. In this study, the serum level of TNF-α ws lso significntly higher in infnts infected with prsites thn those uninfected. In ddition, the level of infected infnts ws significntly lower in the brest-fed group thn tht in the non-brest-fed group. The serum level of TNF-α in the brest-fed group ws lso significntly lower thn tht in the non-brest-fed group with the sme intensity of prsite infections. Tumor necrosis fctor-induced nitric oxide production by mcrophges leds to cytotoxicity of E. histolytic in vitro [31]. The serum levels of TNF-α of mle infnts were significntly lower thn those of femle infnts in the infected brest-fed group. Gender differences exist in the incidence, morbidity, nd mortlity ssocited with diseses with n inflmmtory component [32]. The reltionship between TNF-α levels nd gender will be topic for further studies. There were no significnt differences in IgE levels between prsite-infected boys nd girls in this study, in greement with the previous report [20]. The present study showed positive reltionships between the intensity of infection nd serum levels of IgE nd TNF-α in the brest-fed group. Thus, the serum levels of IgE nd TNF-α my be mrker of the intensity of infection nd n indictor for therpy nd prognosis in infnts with persistent dirrhe. The present study reveled the effect of brest-feeding on lowering the rte nd meliorting the intensity of protozon prsite infections. The spred of brest-feeding by eduction nd encourgement of mothers, with ttention to trnsmission of pthogens such s humn immunodeficiency virus through brest-feeding, cn prevent protozo-cused dirrhe, the min cuse of deth in infnts [33]. ACKNOWLEDGMENTS The uthors would like to thnk Prof. Nbil Shokrny Gbr, Hed of Deprtment of Prsitology, Fculty of Medicine, Mini University, Egypt for his continuous dvice through the technicl procedures. The uthors would lso like to thnk Prof. Reft M. Khlif, Deprtment of Prsitology, Fculty of Medicine, Assuit University, Egypt for his continuous dvice. Conflict of interest We hve no conflict of interest relted with this study. REFERENCES 1. Pierce KK, Kirkptrick BD. Updte on humn infections cused by intestinl protozo. Curr Opin Gstroenterol 2009; 25: Fischer Wlker CL, Perin J, Aryee MJ, Boschi-Pinto C, Blck RE. Dirrhe incidence in low- nd middle-income countries in 1990 nd 2010: systemtic review. BMC Public Helth 2012; 12: Cheng AC, McDonld JR, Thielmn NM. Infectious dirrhe in developed nd developing countries. J Clin Gstroenterol 2005; 39: Speich B, Mrti H, Ame SM, Ali SM, Bogoch II, Utzinger J, Albonico M, Keiser J. Prevlence of intestinl protozo infection mong school-ged children on Pemb Islnd, Tnzni, nd effect of single-dose lbendzole, nitzoxnide nd lbendzolenitzoxnide. Prsit Vectors 2013; 6: Section on Brestfeeding. Brestfeeding nd the use of humn milk. Peditrics 2012; 129: e Korpe PS, Liu Y, Siddique A, Kbir M, Rlston K, M JZ, Hque R, Petri WA Jr. Brest milk prsite-specific ntibodies nd protection from mebisis nd cryptosporidiosis in Bngldeshi infnts: prospective cohort study. Clin Infect Dis. 2013; 56: Tellez A, Winieck-Krusnell J, Pnigu M, Linder E. Antibodies in mother's milk protect children ginst girdisis. Scnd J Infect Dis 2003; 35:

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