1. The Vicious Cycle of Diarrhea and Malnutrition:
Effect on Growth and Development
Cognition, Obesity, Biomarkers, Pathogens
Richard L Guerrant
Aldo AM Lima, Reinaldo Oria,
Jim Roche, Rebecca Scharf, Mark DeBoer, Jim Nataro,
Orleancio Azevedo, Noronha Diniz, Pranay Sinha, et al
Infectious Diseases and International Health
Centers for Global Health
University of Virginia-Federal University of Ceara-ICIDR-NIAID/FIC/FNIH
Charlottesville, VA-Fortaleza, Brazil
The Global Crisis of Childhood Diarrhea
PROGRAMME FOR GLOBAL PAEDIATRIC RESEARCH SYMPOSIUM
Boston - April 30, 2012
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2. Disclosures
Cofounder of AlGlutamine, LLC
Member Probiotics Scientific Advisory Board

3. The Vicious Diarrhea-MN Cycle
Diarrhea
Inc. incidence +
Inc. duration
& severity
Malnutrition
Dec. Wt gain (short)
Dec. Ht gain (long)

4. Malnutrition increases incidence
and duration of diarrhea
>-3 WAZ <-3 WAZ %inc. p value
Episodes/2m 1.9 2.6 37% 0.001
Duration (d) 6.7 11.6 73% 0.004
Total d of D. 12.1 24.2 100% <0.001
•By Wilcoxon rank sum test
•Also valid for moderate MN (<90% HAZ) Schorling JB et al, Int J Epi 19: 728-735, 1990.

5. ECD /Enteric Infections Stunt Growth („60s-‟80s)
•Guatemala
•“Matagram” and dysentery/bpnuem Scrimshaw Taylor Gordon; Mata etal. Am J Clin Nutr24:249, 1971.
•ECD (not fever/pneumonia) stunts Ht 6.5%, irresp of feeding Martorell R et al. Am J Dis Child
129: 1296, 1975.
•Gambia
•ECD (not ARI) impairs Ht & Wt gains Rowland MG et al. Br J Nutr 37: 441, 1977.
•ECD (& ARI) <1yo/nonBF impairs Wt gains Rowland et al. Am J Clin Nutr 47:134, 1988.
•Mexico
•ECD (not ARI) impairs Wt gains to 10% @3yo Condon-Paoloni et l. AJPH 67: 651, 1977.
•Brazil
•ECD impairs Wt (32% @2wks), Ht (41% @ 3.5m) Guerrant et al. JID 148: 986, 1983.
•ECD blunts catchup growth & PD (not fever/pneumonia) stunts Wt & Ht Leslie J, de
Souza MA. Ch 15 in Edge of Development 1996; Schorling, Guerrant Lancet 335: 599, 1990; Moore et al Gastro 139:
156, 2011.
•Bangladesh
•ECD (not ARI) stunts Wt @ 2m & 2/10cm Ht @ 5yo, esp. Shigella, ETEC but irresp of
etiol. Black RE et al, Pediatr 73: 799, 1984.

6. “Matagram” showing that repeated diarrheal and
other illnesses pull children off their growth curves
Mata L. The children of Santa Maria Cauque. MIT Press, Cambridge MA. 1978.

7. Starting ok
WHZ but losing
WAZ at 3-24 months!
HAZ
Victora…Shrimpton,
Pediatrics 125: e473, 2010. Latin America
Africa
Pediatrics 107: e75, 2001. S.Asia

8. Diarrhea ablates catch-up growth
0.5
Weight gain (kg/month)
0.4
0.3
Weight for age <-3
Weight for age >-3
0.2
0.1
0
<15 15-30 31-45 >45
% of Days with Diarrhea
Schorling JB, Guerrant RL. Diarrhea and catch-up growth. Lancet 335: 599-600, 1990;
Petri et al. JCI 118: 1278, 2008; Guerrant et al. Nutr Rev 66: 487, 2008; Checkley et al. IJE 37: 816, 2008.

10. Early childhood diarrhea (0-2 years old) impairs height-for-
age at 2-7 years old (8.2cm)
Moore SR et al, Int J Epi 30: 1457, 2001.

11. Multi-country analysis confirms that
diarrhea increases stunting (HAZ-2 <-2)
Every 5 episodes stunts 13%
(over incidence range of 3.6-13.4 episodes/child-yr!)*
* >5 episodes of diarrhea before 2yo
accounts for 25% of all stunting. Checkley et al, Int‟l J Epi 37: 816-30, 2008.

12. The Vicious Cycle of Diarrhea and Malnutrition:
Effect on Growth and Development
Cognition, Obesity, Biomarkers, Pathogens
•Vicious cycles of the diseases of poverty;
•ECD lasting stunting and cognitive impairment;
•Potential effects of early infections and stunting on
obesity/DM/MetS/CVD;
•Biomarkers/Pathogens-esis/Novel Interventions

13. Infectious Diseases Impair IQ The Economist, July 1, 2010.
When controlling for :
•GDP per capita;
• Education (WB lit.AVED)
•Temperature;
Nutritional deficiencies
correlation w IQ was
lost when ID removed
ie ID, not MN or
poverty may cause
the “Flynn Effect” of
IQ:Development
Eppig C, CL Fincher, R Thornhill.
Proc Biol Sci 277: 3801, 2010.

14. Vicious Cycles of the Diseases of Poverty
Enteric & Other Anti-
Water,
Sanitation
Infections +Diarrhea Microbials;
Vaccines
Environment
Worsened Infection
Intensity & Damage Intestinal Damage
Repeated & & Inflammation
Microbiome Persisting
Impaired Innate & Infections Nutrient
Acquired Mucosal Malabsorption
Defenses &/or Loss
Hs genome
Probiotic
Malnutrition Repair
Nutrients
Microflora
Impaired Vaccine Responses
Cognitive Impairment
Obesity
DM, HT, CVD,
Poverty Met. Synd.
$$Health Care

15. Albendazole Improves Physical and Cognitive
Development (hookworm, Ascaris &Trichuris)
•Kenyan schoolchildren had better Fitness, Growth
(W+L) and Appetite 2-4m after single Albendazole.
Stephenson L, et al. AJTMH 41: 78, 1989; TRSTMH 84: 277, 1990;
J Nutr 123: 1036, 1993.
•Jamaican schoolchildren had better short & long-
term memory (WISC digit span), Fluency and scanning
2 m after Albendazole x3d & arithmetic 10y w HAZ-2.
Nokes C, Grantham McGregor S, Bundy D, et al. Proc R Soc Lond B 247:
77, 1992 ; Chang SM et al J Child Psychol Psychiat 43:775, 2002.

16. Global Burden of Infections/MN:
Decreasing Mortality, but Increasing Morbidity
Of 7.6-10.6m Child Deaths/yr:
Diarrhea specific mortality
25
Mortality
rate per thousand
20
1955-1979
15
YLL 10
5
1982-1990
1990-2000
(Yrs of Life Lost) 0
<1y 1-4y 0-4y
Malnutrition Age group
53%
Diarrhea 15-18% (>3100/d)
Morbidity 6
Episodes per Person Year
5
YLD 178m stunted (HAZ<-2)
4
3
1955-1979
1980-1990
(Yrs Lost to Disability) MN is THE leading risk 2 1990-2000
1
20% of ALL children; factor for health loss! 0
32% DC children! 11-16% of all DALYs ; 0-5m 6-11m 1y 2y 3y 4y
Age group
35% of all <5yo DALYs!*
DALYs (Disability Adjusted Life Yrs)
Black , Bryce et al. Lancet 375: 1969, 2010;
*Black et al Lancet; 371:243, ‟08;
(YLL + YLD) Murray, Lopez Lancet 349: 1436, „97;
367: 1747, „06.
365:1147, 2005.
Kosek et al. Bull WHO 81: 197, 2003; .
Bhutta et al Lancet 371: 417, „08. JHPN 27:319, 2009.
Guerrant et al Tr Parasitol 18: 191, 2002.

17. Diarrhea increases stunting &
Stunting impairs Cognitive
Development & Schooling
Mod-sev HAZ-2yo 25-61% IQ z lower @ 8-11yo
(Philippines Nonverbal Intel. - Guthrie test; p<0.001)
•Both severity and timing dependent; only 1/3 catch up
•Partially reversible
•Compounded with indirect effects on schooling.
30% lower
IQ @ 11yo
Stunting impairs cognition, but schooling still helps
Stunting delays schooling Mendez M & Adair L. J Nutr 129: 1555-62, 1999.

18. Nutrition @ 0-2yo increases IQ and earning
Atole veg/prot suppl (2/4 Guatemalan villages ‟69-‟77)
(163kcal/11.5g protein vs 59kc/0prot “Fresco” -INCAP Martorell, et al)
•10% better Raven IQ
(AFTER adjusting for schooling)
Stein AD et al. Arch Ped Adolesc Med 162: 612-8, 2008.
•46% higher wages @ 25-42yo m
•8-20% higher reading, Raven, schooling f
(But not after 3yo!)
Hoddinott J et al. Lancet 371: 411-6, 2008.

19. Magnitude of lasting disability effects
from early childhood diarrhea (ECD):
1. Growth shortfalls (esp. HAZ-2; 8.2cm by 7yo)
HAZ-2 is the best predictor of human capital!
(schooling, economic productivity, offspring wt!)
Victora C. et al, 5 cohorts (Bz, Guat, India, Philipp, S Africa) Lancet 371: 340, 2008.
2. Fitness impairment (=17% decr. work prod.)
3. Cognitive impairment (c. 10 IQ points)
4. School performance (c. 1 yr)
(increased age at starting school and age-for-grade)
These effects >DOUBLE the global diarrhea DALYs.
Moore et al. Int J Epi 30: 1457, 2001. Guerrant DI et al. Am J Trop Med Hyg 61: 707, 1999.
Niehaus et al Am J Trop Med Hyg 66: 590, 2002. Guerrant et al Tr Parasitol 18: 191, 2002. Lorntz et al PIDJ 2006.

20. Low HAZ-2 correlates with:
•Adult stunting
•Less attained schooling (1y=12-14% lifetime earnings)
•Reduced economic productivity (Monthly income)
•Lower offspring birthwt (in females)
•Hi glucose, BP, lipids (p BMI controlled for,
implicating subsequent wt gains; ?Barker hypothesis;
“Thrifty epigenotype” & genetic „canalization‟)
HAZ-2 is the best predictor of human capital!
(schooling, economic productivity, offspring wt!)
Victora C. et al, 5 cohorts (Bz, Guat, India, Philipp, S Africa) Lancet 371: 340, 2008.

21. The cognitive deficit most impaired with diarrhea
is Semantic Fluency (as in Alzheimer‟s Disease)
(and ApoE4 protects children‟s cognition with heavy diarrhea; “thrift allele”)
125
R2 = 0.135, p=0.012
100
Test of
Non-verbal
Intelligence-III
quotient
75
IQ at
6-9yo
50
0 5 10 15 20 25 30 Guerrant DI et al. Am J Trop Med Hyg 61: 707, 1999. Niehaus et al.
Diarrhea rates at 0-2yo AJTMH 66: 590, 2002. Patrick et al. Child Neuropsych 11: 233, 2005.
Oria R, Patrick P, Lima A, et al Pediatr Res. 57: 310, 2005.

22. Neurocognitive markers of
cortical function (>5yo):
•TONI/Raven Prog. Matrices
•WRAML Verbal learning
•WISC Coding
•NEPSY Fluency (Sem/Ph)
(vs Developmental Psychology;
nature + nurture: Bailey, HOME;
McArthur Language)

23. Most (>75%) of Human Brain Growth & Development
Occurs in the First 2 Years of Life
Human brain Human brain wt/age
growth
Human postnatal visual cortex dendritic tree
birth 15 m 2 yo
+ only 1 opportunity for neuronal connections or „forever lost‟

24. Importance of a Healthy Gut in Early Childhood
To full growth and healthy development:
Key in first 2 years of life, because that‟s when:
1.Synaptic “tracks” are laid;
2.Most vulnerable
Mendez M & Adair L. J Nutr 129: 1555-62, 1999.
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Stein AD et al. Arch Ped Adolesc Med 162: 612-8, 2008.
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Hoddinott J et al. Lancet 371: 411-6, 2008.

25. Might ECD/Stunting also risk Obesity?!
[Bad water colliding with bad diets]
Of 117/532 Adults in Maceio Slum (NE Brazil) who were
stunted:
• 30% (vs 23.6% of nonstunted) were overweight or obese
vs
• 16% (vs 23% of nonstunted) were underweight
?via increased cortisol:insulin or decreased IGF-1 levels
??Transgenerational epigenetic DNA methylation/silencing
Sawaya AL et al. Nutr Rev 62: S127-133, 2004;
Varela-Silva et al. Coll Anthropol 1: 39-46, 2007.

26. Low HAZ-2 correlates with:
•Adult stunting
•Less attained schooling (1y=12-14% lifetime earnings)
•Reduced economic productivity (Monthly income)
•Lower offspring birthwt (in females)
•Hi glucose, BP, lipids (p BMI controlled for,
implicating subsequent wt gains; ?Barker hypothesis;
“Thrifty epigenotype” & genetic „canalization‟)
HAZ-2 is the best predictor of human capital!
(schooling, economic productivity, offspring wt!)
Victora C. et al, 5 cohorts (Bz, Guat, India, Philipp, S Africa) Lancet 371: 340, 2008.

27. Long-term epigenetic effects
& evolution of imprinting
(Dense cytosine methylation of CG dinucleotides silences promoters)
• Heavy LEP promoter methylation occurs during
postzygotic development in mice & humans.
Stoger R. Epigenetics 1: 155, 2006.
Stoger R. BioEssays 30: 156, 2008.
• LEP (and IGF2, IL10, ABCA1, GNASAS, MEG3) methylation greater in
WBC of periconceptually DHW exposed vs sibs.
Tobi, Stein et al. Hum Mol Gen 18: 4046, 2009.

28. Early MN Later Obesity & Heart Disease
• 300k 19yo men more obese if mothers pregnant
in Dutch Hunger Winter of 1944-5.
Ravelli GP et al. NEJM 295: 349, 1976.
Whitelaw NC & Whitelaw E. Hum Mol Genet. 15: R131, 2006.
• 7845 women in DHW Famine (@10-17yo) have
27% more HD.
vanAbeelen A et al. BBC News & Europ Heart J. Aug 25, 2011.
• Early Childhood Stunting (or lo BWt) has 29-65%
greater Abd obesity (waist:hip/eaHAZ-3)
Schroeder, Martorell, Flores. AJEpi 149: 177, 1999.

29. Chronic consequences of enteric infections:
Physical and cognitive development
Margolis „10
Niehaus „02
Pinkerton „11
Eppig „10
Mendez, Adair „99
Moore ‟01 Cognitive
ECD Checkley „08 Early Stein, Martorell „08
Sinha P „11
Impairment
Enteropathy Stunting
(Early Childhood HAZ-2 Van Abeelen ‟11
Diarrhea) Victora ‟08 ; Martins 06;
Sesso 04; Ferreira „09
BMI-2
Preg/LBWt
Grillol 05;
Barker NEJM „05
Adult
HD/HT
Schroeder „99
Ferreira „01, „09
Obesity DM/GiTT
Bhargava „04
Ravelli (DHWinter) „76 Abd (waist:hip) Met. Synd
Barker „86, „88, „90 , ‟92
Fall ‟08 HiLDL; LoHDL
Victora „08
Poor Later
childhood obesity Poor
Later
growth childhood
2h G>140@30yo CHD<64yo Z growth
obesity
1.5k Delhi 8k Norway
BMI
Bhargava „04 Barker „05

30. JID 148: 986, 1983.
CID 28: 966, 1999.

31. Markers and Impact of Environmental Enteropathy
Assessing: Urine Stool Blood
Barrier/Abs Damage: L/M ** Alpha1-AT EndoCAb**
Cr ?Zonulin* Zonulin
?Intestinal FABP* ?Intestinal FABP* Intestinal FABP
Inflammation: MeBlue Microscopy
(Hemoccult)
Lactoferrin (nonBF)**
MPO
Nitric Oxide (ie IL-8 et al
nitrates+nitrites) * ?IL-8 mRNA*
?Calprotectin*
??Calprotectin mRNA* **Documented to
be associated
?Neopterin* with stunting.
?SAA3*
Repair: ?Citrulline* ?Citrulline *Research
(?Reg-1*) questions.
Small Bowel Overgrowth Lactulose Breath Test
Tissue Biopsy +quantitative culture Gut Integrity
METABONOME Meeting-Seattle,
Field History: Hx of ProD >7d** December, 2010

32. Prolonged or Persistent Diarrhea
(ie any diarrhea >7d)
signals heavy diarrhea burdens and
weight and height growth shortfalls;
ie.
provides a “field biomarker” for
impending malnutrition!
Moore, Lima et al. Gastroenterol. 139: 1156, 2010.

33. Emerging causes of Persistent Diarrhea/MN
in Fortaleza, Brazil
Cases Controls
(n=127) (n=331)
Enteroaggregative E. coli
AA probe + 32%* 14%
AA probe - 36* 17
Cryptosporidium 25** 5
Giardia lamblia 21** 8
Ricci et al. Nature S1: 29, 2006
*P<0.05; **P<002 Fang et al J Ped Gastro Nutr 1995
Koopmans et al Ped ID J 16:504, 1997
Lima et al. J Infect Dis 181: 1643, 2000
Steiner T et al. J Infect Dis 177: 88, 1998.

34. Emerging causes of Persistent Diarrhea/MN
in Fortaleza, Brazil
Cases Controls
(n=127) (n=331)
Enteroaggregative E. coli
(-0.76 HAZ; p<0.05; Steiner JID 177: 88,‟98
EAEC w MN in MalED CC Lima „12)
AA probe + 32%* 14%
AA probe - 36* 17
Cryptosporidium 25** 5
(Checkley „97; ‟98; Agnew JID 177: 754,„98)
Giardia lamblia 21** 8
(Newman TMIH 6: 624, ‟01 Sx w MN; PD>AD) Ricci et al. Nature S1: 29, 2006
Fang et al J Ped Gastro Nutr 1995
Koopmans et al Ped ID J 16:504, 1997
Lima et al. J Infect Dis 181: 1643, 2000
*P<0.05; **P<002 Steiner T et al. J Infect Dis 177: 88, 1998.

35. Evidence for lasting disability effects
from early childhood diarrhea/enteric infections*
Growth shortfalls (esp. HAZ-2; 8.2cm by 7yo)
•Crypto Infections increase diarrhea morbidity and nutritional shortfalls to 18m
[Agnew 98; Lima 00; Newman 99]
•Crypto infections +diarrhea = dec. wt gain @1m [Checkley 97]
•Crypto infections <6m/stunted = .95-1.05 cm deficits @1y [Checkley 98]
•EAggEC infections + inflammation = growth shortfalls [Steiner 98]
•Diarrrhea<2yo = 3.6cm stunted @7yo (8.2cm w helminths) [Moore 01;
+ Checkley et al, 08]
Fitness impairment (=17% decr. work prod.)
•Albendazole =7% inc. HST @4m [Stephenson 93]
•Diarrhea or Crypto <2yo = 4-8% dec. HST @4-7yo [Guerrant 99]
•4.3% inc. HST = 16.6% inc. work prod. [Ndamba 93]
Cognitive impairment (c. 10 IQ points)
•Diarrhea <2yo dec. WISC coding/digit @5-9yo [Guerrant 99]
•Diarrhea <2yo dec. TONI @6-10yo [Niehaus 02]
•Giardia or stunting = 4-10 pts dec. WISC-R @9yo [Berkman 02]
School performance (c. 1 yr)))
•Diarrhea <2yo = inc. AASS; AFG [Lorntz 06; Guerrant 02]
* Petri et al JCI 118: 1277-1290, 2008; Guerrant et al Nutr Rev 66: 487-505, 2008.

36. Cryptosporidium, EAEC, Giardia
or Intestinal Inflammation
Stunt Growth and/or Psychomotor Development
•Fecal neopterin (inflammation) in 72 children
followed 15m in rural Gambia had impaired long-term
ht and wt gains (r=-0.29 and -0.36; p<0.001)
[not Giardia or L/M]
Campbell DI et al. J Ped Gastro Nutr 39: 153, 2004.

37. ECD or Giardiasis Stunt Growth and Cognition
•Stunting or Giardia Peru <2yo WISC-R IQ @ 9yo
Berkman DS et al. Lancet 359: 564, 2002.
•Of 160 Children <5yo in Turkey, those w Giardia were stunted
(OR=7.7) and had psychomotor impairment (OR=2.7)
Simsek Z et al. J Trop Pediatr 50: 90, 2004.
•Giardia Egypt <4yo had cognitive development if E4-
Yahya RS et al. Internet J Parasit Dis 4:1, 2009.
•Heavy ECD or Giardia <2yo Sem. Flu @ 6-12yo E4-
Newman TMIH 6: 624, „01 PD; Patrick PD et al. Child Neuropsych 11: 1-12, 2005
Oria R et al. Ped Res 57: 310, „05; Bz J Biol Res 43: 249, 2010.
•Giardia 6-13yo Iran had WISC Fluency, Span (indep of WAZ)
Partovi F et al. Iran JPH 36: 73, 2007.
•Asxy Giardia Salvador <4yo have stunting 1y later
Prado MS et al. Parasitol 131: 51, 2005.
•Sx Giardia S India predict wasting and cognition@ 3yo
Ajjampur SSA et al. Trop Paediatr 31: 203, 2011.
Guerrant, Oria, Moore, Scharf, Lima “ 201, 2011.

38. Enteroaggregative E. coli (EAEC) causes
Persistent Diarrhea, Intestinal Inflammation, Growth Shortfalls
•EAEC is associated with 36% of PD; 30-74% w HIV
•EAEC-PD has elevated fecal LF, IL-8, IL-1 [Fli-C; TLR5]
•EAEC-no D. also had elevated LF & Growth Shortfalls
Steiner T et al JID 177: 88, 1998; Wanke et al JID 178: 185, 1998;
Samie A et al. 77: 142, 2007; Steiner T et al JCI 105: 1769, 2000.
Nataro et al and Steiner et al CID: 2006.
EAEC Turista (26%)
Only with IL-8 -251 AA or AT Polymorphism
•0/23 w EAEC Diarrhea had TT vs 9-18% of 46 others without Sx
or without EAEC (OR = 208; p<0.01)
•Also those with -251 AA had greater fecal IL-8 (vs AT or TT;
p<0.01) Jiang ZD et al. JID 188: 506, 2003.

39. MalED Case-Control Site, Fortaleza, Ceará, Brazil
•Serves 313
km2 ; 13km
from UPC
•21k children IPREDE Nutrition Clinic, Fortaleza
•2000 new
children/yr
•30% of <3yo
have HAZ <-2
n=380
children
enrolled
Aldo Lima, Alvaro Madeiro, Sulivan Mota, et al. 2010-12.

40. Brazil case control: EAEC with MN
Microorganism Control Cases * p values Total
N (%) N (%) N (%)
EPEC 3/62 (4.8) 1/88 (1.1) ns 4/150 (2.7)
aEPEC 1/62 (1.6) 5/88 (5.7) ns 6/150 (4)
EAEC 4/62 (6.5) 22/88 (25) p = 0.0147 26/150 (17.3)
EHEC 3/62 (4.8) 3/88 (3.4) ns 6/150 (4)
EIEC 5/62 (8.1) 8/88 (9.1) ns 13/150 (8.7)
ETEC 2/62 (3.2) 4/88 (4.5) ns 6/150 (4)
Campylobacter 14/70 (20) 12/104 (11.5) ns 26/174 (14.9)
Adenovirus 0/42 (0) 1/72 (1.4) ns 1/114 (0.9)
Astrovirus 0/42 (0) 1/72 (1.4) ns 1/114 (0.9)
E. histolytica 2/38 (5.3) 2/63 (3.2) ns 4/101 (4)
Giardia 7/38 (18.4) 12/63 (19) ns 19/101 (18.8)
Cryptosporidium 2/38 (5.3) 1/63 (1.6) ns 3/101 (3)
EPEC: enteropathogenic E. coli; EHEC: enterohemorragic E. coli; EAEC: enteroadherence E. coli (aaiC + aatA);
EIEC: enteroinvasive E. coli.
Case: child <-2 WAZ; and control: >-1 WAZ; * Fisher‟s exact test. Aldo Lima, A Havt et al 2011.

41. Cryptosporidiosis impairs growth:
•Even “Asyx” (63 vs 37%) 162-342g less @3m
•Stunting (persisting @ 1y) if:
•Infection @ <5m (Agnew <1y 1-2 Z @ 2yo)
•Stunted @ time of infection
•Also in Africa (Molback) if <1yo, no catchup
• And Asia (Ajjampur) Checkley et al. Am J Epi 145: 156, 1997;
multiple Crypto common “ “ 148: 497, 1998;
Agnew et al. JID 177: 754, 1998;
and lower HAZ-2 Molback et al. Am J Clin Nutr. 65: 149, 1997;
Ajjampur et al. AJTMH 83: 1110, 2010.

42. Murine model of weanling malnutrition
& Cryptosporidium infection
 C57BL/6J mice at 7 days
old. Note the difference
in the body weight between
the nourished and
malnourished mice.
4h of separation
8h of separation
12h of separation
C57BL/6J Wild types
4 5 6 = Crypto infection 14
Age (days)

43. Cryptosporidium infection causes Malnutrition
ie. MN is an ID AND
MN worsens Crypto infection
275 NC (n=11)
250 NI 106 (n=16)
% initial weight (g)
MC (n=10)
225 41%
loss MI 105 (n=14)
200 MI 106 (n=16)
175 further 48% loss
150
125
**
100
**p<0.01 vs all groups by
6 7 8 9 10 11 12 13 14 ANOVA‟s Bonferroni test
Age (days)
Coutinho, Oria, Meton, Sevilleja, et al. J Parasitol 94: 1225, 2008.

44. Synergistic effects of malnutrition and
Cryptosporidium infection on ileal architecture
(H&E; 10X; at 14do; 8d p infection)
Nourished Non-infected Malnourished Non-infected
Nourished Infected Malnourished Infected
Coutinho, Oria, Meton, Sevilleja, et al. J Parasitol 94: 1225, 2008.

45. Malnutrition is associated with a 1 to 2.3 log (ie 10 to
200-fold) heavier infection by fecal shedding or in colon
or ileum in 107 Cryptosporidium-infected mice
at peak infection (d8)
2.50
2.00
log Mean (# of parasites / mg of ileal tissue)
log Mean (# of parasites / mg of stool)
2.00
1.50
1.50
1.00
1.00
log Mean (# of parasites / mg of colonic tissue)
2.00
0.50
0.50 1.50
0.00
1.00 -0.50
0.00
MN (n=12) N (n=14) MN (n=6) N (n=3)
Group Group
0.50
Error bars: +/- 1 SE Error bars: +/- 1 SE
Fecal Shedding 0.00
Parasites/gm ileum
(MN = 1.55 vs N = 0.54 MN (n=7) N (n=3)
(MN = 2.02 ± 0.11 vs N = -0.25 ± 0.13,
log/mg; N=26 mice in 3 paired Group
Error bars: +/- 1 SE
n=9 mice, p < 0.001)
experiments; p=0.015)
Parasites/gm colon
(MN = 1.79 vs N = -0.14, n=10 mice, p= 0.001)
Detected by Real-time PCR Coutinho, Oria, Meton, Sevilleja, et al. J Parasitol 94: 1225, 2008.

46. Cryptosporidium causes and worsens MN
Unexcysted oocysts in weaned, 2% protein MN mice
N, Uninfected P<0.03
14%
P<0.05
MN MN-Infected
N, infected
MN, Uninfected
23%
P<0.03
MN, infected P=0.029
Infection @ d58
Costa L, Noronha, Roche, et al. JID 205: 1464, 2012.

47. “Peace Corps Gut”
Normal PCV w Diarrhea and Malabsorption (d-xylose & B12)
40% (of 114) had d-xylose <5g
52% (of 23) had low Schilling B12
Lindenbaum et al. AIM 65: 1201, 1966.
Bx nl in 2/17 (12%); 8 had mild ;7 moderate “jejunitis”

48. Candidates to help “repave”
the small bowel absorptive
tennis court
(every 3 days!)
Normal HIV+ Patient with Cryptosporidium infection
Yamada et al. (1995) Conlon et al. (1984)
Zinc, Vitamin A, Glutamine, Alanylglutamine, Arginine, ?ApoE
peptides, targeted antimicrobials, adjuvants, vaccines
Carneiro, Bushen, Brito, Lima, Guerrant. Curr. Infect Dis Rep. 5: 114, 2003.
Bushen et al. CID 38: 1764, 2004; Lima N et al. J Pediatr Gastroent Nutr 44: 365, 2007.
Ueno… Moore. Am J Physiol GI Liver Physiol 301: 2011. Castro I et al Nutrition, 2011.

49. Vicious Cycles of the Diseases of Poverty
Enteric & Other Anti-
Water,
Sanitation
Infections +Diarrhea Microbials;
Vaccines
Environment
Worsened Infection
Intensity & Damage Intestinal Damage
Repeated & & Inflammation
Microbiome Persisting
Impaired Innate & Infections Nutrient
Acquired Mucosal Malabsorption
Defenses &/or Loss
Hs genome
Probiotic
Malnutrition Repair
Nutrients
Microflora
Impaired Vaccine Responses
Cognitive Impairment
Obesity
DM, HT, CVD,
Poverty Met. Synd.
$$Health Care

50. Diarrhea (Ac, Pro, PD) predisposes to
growth decrements (WAZ or HAZ)
*
Moore, Lima et al. Gastroenterol. 139: 1156, 2010.

51. Glutamine and Ala-Gln ORRT
improve the repair of intestinal epithelium
in IEC6 cells and in
children and HIV+ pts with diarrhea.
Control with Glutamine with Ala-Gln
Brito, Carneiro, Lima, Guerrant. 2004.
Carneiro, Bushen, Brito, Lima, Guerrant. Curr. Infect Dis Rep. 5: 114, 2003.
Bushen et al. CID 38: 1764, 2004; Lima N et al. J Pediatr Gastroent Nutr 44: 365, 2007.
Ueno… Moore. Am J Physiol GI Liver Physiol 301: 2011.

52. Diarrhea impairs Cognitive
Development & Growth
independently
ECD0-2, HAZ2 or WAZ2 predicts TONI & Coding @ 6-12yo
(Brazil cohort of 131 followed from birth; UV p<0.05)
•Excluding household income, maternal education,
child sex, birth wt, breastfeeding
•ECD remained a predictor of IQ even after adjusting
for HAZ and WAZ (multivariate p< 0.006-0.029)
Pinkerton, Oria, Lima, Patrick et al. 2012.

53. Prolonged and persistent diarrhea account for only
100%
17% of episodes, yet half of all diarrhea days
AND predict heavy diarrhea burdens and growth shortfalls!
83%
Acute (<7d)
75% Prolonged (7-13d)
Persistent (≥14d)
% of diarrhea
morbidity
50%
50%
25% 25%
25%
12%
5%
0%
By episodes By days
Moore, Lima et al. Gastroenterol. 139: 1156, 2010.

54. Proposed model for Ala-Gln coupling to EGFR signal
transduction during enteric infections and malnutrition.
Moore S, Polk B et al. 2009.

55. AlaGln improves Wt, morphometry, apoptosis &
proliferation in MN weaned C57Bl/6 mice
Ueno… Moore.
Am J Physiol
GI Liver
Physiol 301:
2011.

56. AlaGln improves barrier function (lactulose
excretion) and weight gains in malnourished
children in Brazil
* p < 0.05
0.4 A *
Peso-para-Estatura
0.3
Cumulativo
WHZ
Escore z
Gain
0.2
0.1
0.0
Glicina Alanil-Glutamina
Lima N et al. J Pediatr Gastroent Nutr 44: 365, 2007.

57. Gln or Ala-Gln enhance absorption
(even with Crypto infections when Glucose fails)
3 3
Ringers Control Ringers Control
10 mM Gln 10 mM Gln
10 mM AlaGln 2 10 mM AlaGln
2
JnetNa
JnetNa
1
1
0
0 n=9 n=9 n=6
n=18 n=18 n=6 Cryptosporidium
Control Infected
Alanyl-glutamine compares favorably to glutamine
in Control and Crypto-infected calves in Ussing
Chambers in driving net sodium absorption
Blikslager et al, Am J Physiol 281: G645, 2001.

58. Another vicious cycle: Barrier disruption & Inflammation
TJ (TNF, MLCK)
(lo cap; nonselect)
Claudin, Occludin,
ZO1, actin,
myosin
AJ (Th2, IL13)
(hi cap; ionselect)
E-cadherin, Lunn P. Proc Nutr Soc
abCatenin,
myosin
59: 147, 2000.
Turner JR Nature Rev,
Immunol 9: 799, 2009.
Salazar-Lindo E....
D Brewster…
A Fasano....P Tarr.
JPGN 39: S662, 2004.
Sears C. Clin Micro
Rev 22: 349, 2009.

59. Urine Metabonome in MN vs Control Children
showing increased NAG, DMA, DMG; less Mannitiol
(by NMRS and PLS-DA coefficient plot)
Projection to Latent Structure-Discriminant Analysis
Cases vs
Control s
p=0.0248
n=75
CA2-3
Swann J,
Lima AAM,
et al. 2011.

60. Enteroaggregative E. coli (EAEC)
Infection Impairs Growth
& MN worsens infection
DMEM / HS (Nourished Control) n=11
EAEC-JM221 (Nourished infected) n=12
EAEC-JM221 (Malnourished infected) n=7 EAEC
29% less
p<0.03 p d8
Further 32% less
with MN
P<0.02
**
Peak shedding = 102-2.8 Nourished/ 103.1-7.5 Malnourished
Roche, Cabel,
Sevilleja,
Nataro et al,
JID 202: 506,
2010.

61. Induces
Glutamine HSP70, which:
•Attenuates iNOS, IL-6, IL-8
(in rat CP bypass)
•Protects against neurodegeneration
(in spinocerebellar degeneration, SCA-1 mice)
Hayashi Y et al. Circ. 106: 2601, 2002.
Cummings CJ et al. Hum Mol Genet 10: 1511, 2001.

62. Cryptosporidium and Giardia genotypes in
Brazil and South Africa
•C. hominis predominates in NE Brazil with:
greater shedding, lactoferrin & growth shortfalls
Bushen, Lima et al. TRSTMH, 2006.
•C. hominis predominates in Limpopo, S. Africa;
assoc. w diarrhea (57%); lactoferrin (59%)
Samie et al. Expt Parasitol 114: 314, 2006.
•74% of Giardia are Assemblage B, with
greater shedding (3.6 v 1.4 x 105/ml); lactoferrin
(especially with first infection; 74 40 20%; also longer)
Kohli, Lima et al., TRSTMH 102: 718-725, 2008.

63. Conclusions (re Crypto and MN)
•Crypto infections, +diarrhea, ProD or PD impair growth in
young children (or mice);
•Likely via disrupted intestinal mucosal function (L/M; V/C);
•MN worsens Crypto (in stunted children or MN mice), thus
completing the vicious cycle (that also impairs cognitive
development, with an “Alzheimer-like” deficit).
•ApoE4 protects against these deficits only in children with
heavy diarrhea burdens.
•ApoE4/4 targeted transgenic Eko mice grow better and have
less barrier disruption & infection.
•Arginine, like ApoE4, protects mice and may protect children
from the vicious diarrhea-MN cycle.

64. Disruption of Intestinal Barrier Function in Children
with Acute and Persistent Diarrhea in Fortaleza, Brazil
0.8
**
0.7
0.6
*
0.5
Mean
Lactulose:
Mannitol 0.4
Excretion
Ratios 0.3
0.2
0.1
0
Control AD PD
n=20 n=18 n=2
[vs controls: *p=0.001; **p<0.001]
Guerrant, Lima, Barboza, et al Adv Exp Med Biol 473:103-11, 1999.
Barboza et al BrasJ Med Biol Res 32:1499-1504, 1999.

65. EndoCAb correlates with disrupted intestinal
barrier function and with stunted growth
Campbell DI, Elia M, Lunn PG. J Nutr 133:1237, 2003.
Petri, Ahmed, Haque et al 2010.

66. Enteroaggregative E. coli (EAEC) causes
Persistent Diarrhea, Intestinal Inflammation, Growth Shortfalls and
is the Leading Bacterial Enteropathogen in US
•EAEC is associated with 36% of PD; 30-74% w HIV
•EAEC-PD has elevated fecal LF, IL-8, IL-1
•EAEC-no D. also had elevated LF & Growth Shortfalls
•EAEC filtrates induce IL-8 release in Caco-2 cells.
•EAEC Fli-C (unique flagellin) cloned/sequenced is
responsible for the IL-8 release.
•EAEC signif assoc with 4.5% diarrhea in Baltimore
and New Haven (vs 2-3% Cj, Salm; 10-12% Rota, Noro)
Steiner T et al JID 177: 88, 1998; Wanke et al JID 178: 185, 1998;
Samie A et al. 77: 142, 2007; Steiner T et al JCI 105: 1769, 2000.
Nataro et al and Steiner et al CID: 2006.

67. HAZ correlates with Grades and Rank
(among 242 college schoolgirls in S. India)
r2=.037 r2=.030
p =.003 p =.007
(Independent of also significant # siblings, paternal education.)
Pranay Sinha, B.S. Ramakrishna, et al. August, 2011.

68. Infant MN CVD
Barker Hypothesis
Barker D. et al. Lancet 1986, „89, ‟90.
Hertfordshire & Southampton, UK

69. “Thrifty epigenotype”
Candidate genes for environmental „canalization‟
Stoger R. BioEssays 30: 156, 2008.

70. Infants with ProD are high risk
for future episodes of PD
(+) Prolonged Diarrhea
prior to age 1 year
*
% of children
with p=0.002
Log-rank test
Persistent
Diarrhea
No Prolonged Diarrhea
prior to age 1 year
Age (months)
Moore, Lima et al. Gastroenterol. 139: 1156, 2010.

71. Cryptosporidium: A Leading cause of
Prolonged and Persistent Diarrhea &
Growth Shortfalls
% w Crypto P (vs no D)*
•Persistent Diarrhea (n=71) 23.9%* 0.0002
•Prolonged Diarrhea (n=98) 12.2%* 0.018
•Acute Diarrhea (n=402) 11.4%* 0.0043
•No Diarrhea (n=289) 5.2% -
Also Giardia with 20.5% of 88 with PD (p=0.0003); and fecal WBC or LF in 43-77% (p<0.02).
Lima AAM et al. JID 181: 1643, 2000.
Moore S et al. Gastroenterol 139: 1156, 2010.

72. Long-term Consequences of
Cryptosporidium Infection
•Malnutrition Sallon ‟88; Sarabia-Arce ‟90; Checkley ‟99; (Peru)
Lima ‟92; (Brazil); Molback ‟93; ‟97; (G-Bissau)
•Even Asxy Crypto Stunting Checkley ‟97; (Peru)
C. hominis > parvum, c 2x shedding Bushen ‟07 (Brazil))
(3.5x106); LF; longer growth delay
•Early Diarrhea or Crypto DI Guerrant et al „99
(Brazil))
Fitness + Cognitive
impairment 4-7y later
•Reactive Arthritis/Reiter‟s Hay ‟87; Ozgul ‟99; Shepherd
‟89; Cron ‟95; Hunter „04
Chalmers & Davies Expt Parasitol 124: 138, 2010.

73. ApoE4 Upregulates an
Arginine-selective Cat-1 Transporter
•Transgenic mice expressing the human APOE4
allele have expression of mRNA for the
constituitive Arg selective y+ transporter (Cat1) in
microglia.
•Hence NO (not iNOS) and cell proliferation.
Colton, et al. Neurobiol Aging 23: 777, 2002
Czapiga & Colton. J Neuroimmunol. 134: 44, 2003.

74. Arginine improves wt gain & reduces ileal
Cryptosporidium infection by over 10-fold by both iNOS
& arginase-dependent pathways
Oocyst shedding in Ileum
200000
* p < 0.05
175000
* p < 0.05
150000
Oocysts per mg of tissue
* p < 0.05
125000
100000
75000
50000
25000
0
PBS Arg 200mM Arg 100mM Arg 200mM + Arg 200mM +
L-NAME 20mM BEC 2.2mM
L-NAME = NG-nitroarginine methyl ester (blocks iNOS);
BEC = S-(2-boronoethyl)-L- cysteine (blocks Arginase) Castro I. et al. Nutrition, 2012.

75. MN-Infected ApoE4/4ki Mice have better growth and
Villus/Crypt Architecture, and shed less Cryptosporidia
Post-infection
110
% of Weight
105
* # ApoE 4/4 MNI (n=16)
WT MNI (n=13)
100 ApoE KO MNI (n=14)
95
0 1 2 3 4 5 6 7
Days
* p<0.05 ApoE TR 4/4 MNI vs Wild type MNI
# p<0.0001 ApoE TR 4/4 MNI vs ApoE KO MNI
Shedding
p<0.05
10 6 p<0.05
Wild type MNI (n=12)
p<0.05 ApoE KO MNI (n=12)
p<0.05
nº parasites/mg of stool
10 5 ApoE 3/3 TR MNI (n=8)
p=0.05
ApoE 4/4 TR MNI (n=17)
10 4
10 3
10 2
1 3 5 7
Groups
Apoe4 mice had no shedding on day 7 De Azevedo, Oria, et al. 2012.

76. Recurrent diarrhea impairs height
and weight gains
Ht gain (cm) Wt gain (kg)
No recurrent diarrhea 1.74 cm 0.44 kg
Recurrent diarrhea* 1.38 cm** 0.25 kg***
* >30% prevalence over subsequent 2m
** 21% less; p=0.1
*** 43% less; p=0.01
Schorling & Guerrant. Diarrhea and catch-up growth.
Lancet 335: 599-600, 1990.