During the 20th century, significant strides were made in curtailing the burden of childhood diarrhea, including advances in vaccine research, the advent of antibiotics, improved water and sanitation, and expanded access to health information across the globe. Despite this progress, today diarrhea ranks second only to pneumonia as a leading cause of mortality in children under five years, with a disproportionate burden of 90% of diarrheal deaths in South Asia and Sub-Saharan Africa. Additionally, substantial morbidity due to diarrhea persists in young children, with more than 45 million disability-adjusted life years (DALYs) lost due to diarrhea in 2015. Long-term consequences of childhood diarrhea include undernutrition, impaired gut function, altered gut microbiota, and compromised cognitive development. The 21st century presents an opportunity to eliminate the health disparity affecting millions of children suffering disproportionately from preventable diarrheal diseases. Recent advances in molecular laboratory technology have enabled detailed assessment of diarrheal burden and etiology, illuminating the highest burden pathogens for focused interventions. Among the top diarrheal pathogens, rotavirus (RV) is the leading cause of diarrhea-attributable death in the first year of life. While we have vaccines against RV, these vaccines consistently underperform in low and middle-income countries (LMICs) with efficacy of 18% to 61% compared to > 85% efficacy in high income countries. Reasons for rotavirus vaccine underperformance remain unclear, and no vaccines are available for other high burden diarrheal pathogens. This requires consideration of complementary and alternative interventions for diarrhea prevention. To assess factors related to rotavirus vaccine performance, we enrolled a 700-infant birth cohort in an urban slum of Dhaka, Bangladesh, in the Performance of Rotavirus and Oral Polio Vaccines in Developing Countries (PROVIDE) study: a randomized controlled trial of a 2-dose monovalent oral rotavirus vaccine (RV1). With a primary outcome of any rotavirus diarrhea (RVD) post-vaccination to one year, we conducted biweekly home-based diarrhea surveillance with rotavirus antigen detection in diarrheal stools by ELISA. We found RV1 efficacy of 51% (95% CI 33.8–63.7) in per protocol analysis. Importantly, among 12 explanatory variables tested for association with RVD, serum zinc concentration (SZC) in infants at week 18 associated with risk of RVD up to one year (OR 0.77, 95% CI 0.66–0.91), independent of vaccination status. This finding led to broader investigation of the relationship between zinc status and diarrhea in the PROVIDE cohort. Among 577 PROVIDE infants, 16.5% were zinc deficient at week 18 (SZC < 65μg/dL). By logistic regression, zinc deficient infants had increased odds of diarrhea in the first year of life compared to zinc replete infants (OR 2.76, 95% CI 1.08–7.04), and they were nearly 4 times more likely to have diarrhea of viral etiology (OR 3.94, 95% CI 1.55–10.03). Furthermore, in Kaplan Meier analysis we found a strong correlation between zinc deficiency and time to first episode of viral diarrhea (median survival 27 vs 33 weeks in zinc deficient vs non-deficient infants, p Our results indicate further consideration of zinc as a critical and modifiable co-factor in ameliorating the burden of childhood viral diarrhea. Carefully designed trials of zinc supplementation interventions could determine whether zinc may fill the gap in protection against childhood viral diarrhea, and inquiries into the zinc-diarrhea molecular pathway could elucidate mechanisms for focused development of future interventions.

This dissertation assesses the factors that lead to the emergence of infectious diseases in children, particularly the emergence of multidrug-resistant organisms (MDROs) and diarrheal pathogens in vulnerable pediatric populations. It includes three manuscripts. The initial study is a systematic review that summarized the role of antibiotic exposure on the acquisition of MDROs in children. Twenty-nine studies met the inclusion criteria and a positive association between prior antibiotic use and subsequent colonization or infection with an MDRO was identified in most studies. There were wide variations among study sites, populations, and definitions of antibiotic use and MDROs. Therefore, limited inferences could be made on which components of antibiotic exposure have the greatest impact on MDRO development. The second analysis examines the relationship between prior stay at a pediatric long-term care (LTC) facility and infection with an MDRO among hospitalized children. This study included 2,945 infections in 258,664 pediatric admissions from 2006 through 2016. At least 1 MDRO was identified in 10% of infections. Of the 1,198 children who had previously resided in a pediatric LTC facility, only 1 child (0.08%) had an MDRO infection. However, prior receipt of pediatric LTC was associated with an increased likelihood of infection (OR 2.4, CI95 1.66 – 3.43), C. difficile infection (OR 2.57, CI95 1.26 – 5.25), days of antibiotic use (OR 1.01, CI95 1.01 – 1.02), length of stay (OR 1.01, CI95 1.01 – 1.01), and death (OR 4.38, CI95 2.93 – 6.55). The concluding study evaluates the association between animals living in or near the home and diarrheal disease in children. This research is a secondary analysis of the Global Enteric Multicenter Study case control study, which investigated the epidemiology of diarrheal illness in children <5 in sub-Saharan Africa and south Asia. Of 9,439 cases and 13,128 controls, 87% had ≥1 animal in their home. In a multivariable analysis adjusting for exclusive breastfeeding, water source, sanitation facility, number of children <5 years in the household, and wealth index, any animal on a child’s compound decreased the odds of diarrhea by 33% (aOR 0.66, CI95 0.59 – 0.74). However, children with diarrhea who had an animal present were not more likely to have a positive stool culture. Overall, the three studies provide a thorough analysis of several factors associated with the infectious disease emergence in children, particularly as related to MDROs and diarrheal disease. Environmental characteristics, including antibiotic use and interaction with animals, were shown to be important factors for emergent infectious disease across diverse settings. The development of pediatric infection prevention interventions should take into consideration environmental risk factors in order to effectively mitigate the risks posed infectious disease emergence.

Copy of one of the author's previously published articles inserted. / Bibliography: leaves 357-404. / 405 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes a methodology for the detailed evaluation of the processes involved in the assessment of recombinant orally administrable vaccines against mucosal pathogens (a bivalent vaccine against diarrhoeal disease in this case) / Thesis (M.D.)--University of Adelaide, Dept. of Medicine 1991

The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world. In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients. Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

Bakgrund: Patienter inlagda på sjukhus behöver näringstillförsel för att kunna återhämta sig. Enteral nutrition är troligen att föredra framför parenteral nutrition, och bör sättas in så tidigt som möjligt. Enteral nutrition kan administreras på olika sätt. Den kan ges intermittent och innehålla nattvila, eller ges kontinuerligt för att undvika plötsliga sänkningar i blodsockernivåer. En oönskad komplikation är lösa avföringar hos patienten. Frågan är om administrationsformen av enteral nutrition kan påverka frekvensen av diarré. Syfte: Att med hjälp av en registerstudie jämföra antal lösa avföringar per dygn mellan två olika administrationsformer av enteral nutrition: intermittent med nattvila jämfört med kontinuerlig tillförsel över hela dygnet. Metod: En kvantitativ retrospektiv registerstudie har genomförts på totalt 50 intensivvårdspatienter med traumatisk skallskada vid Norrlands Universitetssjukhus. Journaler från 2007 till 2012 har använts i studien. Resultat: Ingen signifikant skillnad kunde påvisas mellan grupperna i antal lösa avföringar per dygn, p=0.5. Däremot visade denna studie att de patienter som fick intermittent enteral nutrition fick signifikant större mängd enteral nutrition (699±249 ml) per dygn jämfört med patienter som fick sin enterala nutrition kontinuerligt (505±278 ml/dygn), p = 0.008. Slutsats: Resultatet från studien visar att antalet lösa avföringar inte verkar bero på administreringsformen av enteral nutrition. Mängden tillförd enteral nutrition skiljer sig dock statistiskt signifikant varav betydelsen av det borde studeras vidare för att avgöra vilken administreringsform som är att föredra. / Background: Patients admitted to hospital need nutrition to recover. Enteral nutrition is probably preferable to parenteral nutrition should be initiated as early as possible. Enteral nutrition may be administered in various ways, it can be intermittent and include night rest, or given continuously to avoid abrupt reductions in blood sugar levels. An unwanted complication is loose stools of the patient. The question is whether the form of administration of enteral nutrition can affect the frequency of diarrhea. Aim: With the help of a retrospective registry study comparing number of loose stools per day between two different forms of administration of enteral nutrition: intermittent with night sleep compared with continuous supply throughout the day. Method: A quantitative retrospective registry study has been carried out on a total of 50 ICU patients with traumatic head injury at Norrlands University Hospital. Records from 2007 to 2012 were used in the study. Results: No significant difference was detected between the groups in the number of diarrhea per day, p = 0.5. However, this study demonstrated that patients receiving intermittent enteral nutrition was significantly greater amount of enteral nutrition (699 ± 249 ml) per day compared with patients who received their enteral nutrition continuously (505 ± 278 mL / day), p = 0008. Conclusion: The results from the study show that the number of loose stools do not seem to depend on the form of administration of enteral nutrition. Quantities of enteral nutrition differ statistically significantly significance of which it should be further studied to determine which form of administration is preferred.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

In der Veterinärmedizin verursachen Rotaviren als Jungtiererkrankung vor allem in der Nutztierpraxis hohe ökonomische Verluste. Über die Prävalenz von Rotavirusinfektionen bei Hunden und Katzen ist sehr wenig bekannt, obwohl von den in der Literatur als wechselseitig zwischen Mensch und Tier übertragbaren Viren ein nicht zu unterschätzendes Risiko ausgehen kann. Zunächst wurden retrospektiv Prävalenzdaten über den Nachweis von Rotaviren bei Hunden und Katzen mit Durchfall im Vergleich zu Coronaviren und Parvoviren erhoben. Dazu wurden Kotproben von 2055 Hunden und 1481 Katzen quantitativ auf das Vorhandensein von Rota-, Corona- und Parvovirus untersucht. Desweiteren wurden Aspekte der geographischen Verteilung, der Altersverteilung, mögliche Rasseprädispositionen und das Auftreten saisonaler Erkrankungsgipfel untersucht und ausgewertet. Für Rotavirusinfektionen beträgt die statistische Prävalenz 7% bei Hunden und 8% bei Katzen. Bei Hunden und Katzen konnten signifikant häufiger Dreifachinfektionen nachgewiesen werden. Bei einer Infektion mit Rota- und Coronavirus liegt beim Hund zu 100% auch eine Infektion mit Parvovirus vor. Zweifachinfektionen kamen weniger häufig vor als Monoinfektionen. Alle drei Virusinfektionen kamen bei Hunden statistisch signifikant häufiger in der Altersgruppe ≤ 1 Jahr vor. Ein statistisch signifikant häufiger Rotavirusnachweis konnte bei der Katzenrasse Siam nachgewiesen werden, während keine Hunderasse besonders hervortrat. Im Postleitzahlengebiet 3 konnten im Beobachtungszeitraum von 2000 bis 2006 statistisch signifikant häufiger Rotavirusinfektionen bei Hunden nachvollzogen werden. Es konnte sowohl für Hunde, als auch für Katzen der Trend belegt werden, dass bei steigenden Lufttemperaturen, die Anzahl der Rotavirusinfektionen sinkt. Es kann somit von einer bedingten Saisonalität ausgegangen werden. Im zweiten Teil der Arbeit wurde die Empfänglichkeit von Rotaviren gegenüber kommerziell erhältlichem Typ I Interferon (rFeIFN-ω) in vitro getestet. Zunächst wurde zum Nachweis der Aktivität der Typ I Interferone (rFeIFN-ω, rBoIFN-α, rHuIFN-α) die Expression des Mx Proteins auf Zelllinien felinen, caninen, bovinen und humanen Ursprungs, sowie auf Affenzelllinien untersucht. Es konnte gezeigt werden, dass rBoIFN-α ausschließlich auf Zellen bovinen Ursprungs eine konzentrationsabhängige Expression des Mx Proteins induziert. Das rFeIFN-ω induziert auf Zellen felinen und bei höheren Konzentrationen auch auf Zellen caninen Ursprungs die Expression des Mx Proteins. Das rHuIFN-α zeigt eine konzentrationsabhängige Induktion des Mx Proteins in Zellen humanen, caninen, felinen und bovinen Ursprunges, sowie in Affenzelllinien. Somit konnte in vitro eine Kreuz-Speziesspezifität für rekombinantes humanes Interferon nachgewiesen werden. Zum Nachweis einer immunmodulatorischen Wirkung wurde die Expression der MHC I Oberflächenrezeptoren nach Behandlung mit rFeIFN-ω und rHuIFN-α untersucht. Die Behandlung mit rFeIFN-ω führte ausschließlich in felinen Zellen zu einer konzentrationsabhängigen signifikanten Erhöhung der Rezeptordichte. Die Behandlung mit rHuIFN-α führte zu einer konzentrationsabhängigen signifikanten Erhöhung der Rezeptordichte auf felinen Zellen und in der Affenzelllinie MA104. Die Empfänglichkeit von Rotaviren gegenüber rFeIFN-ω wurde auf der embryonalen felinen Fibroblastenzelllinie (KE-R) und auf der embryonalen felinen Gehirnzelllinine (KG-R) unter steigender Interferonkonzentration (101-104 Einheiten/ml) untersucht. Beide Zelllinien zeigten eine deutliche Reduktion der infizierten Zellen bei steigender Interferonkonzentration. Die antivirale Wirkung war in KE-R Zellen deutlicher ausgeprägt. Dort konnten bereits bei einer Interferonkonzentration von 103 Einheiten/ml keine sichtbar infizierten Zellen mehr nachgewiesen werden, während KG-R Zellen erst bei einer Konzentration von 104 Einheiten/ml keine sichtbar infizierten Zellen mehr nachzuweisen waren. Abschließend wird deutlich, dass Infektionen mit Rotaviren ein vielmals vernachlässigtes Problem in der Veterinärmedizin darstellt, vor allem, wenn man von einer Vergesellschaftung mit den für Hund und Katze pathogenen Viren Corona- und Parvovirus ausgeht. Mit dem rFeIFN-ω steht in vitro eine wirksame antivirale Substanz gegen Rotavirusinfektionen zur Verfügung.

This cross-sectional, community-based study was designed to compare the health outcomes of 2 - 5 year-old children in different types of preschools. The Integrated Child Development Services (ICDS), run by the government of India, created a system of preschools, called anganwadis, to combat malnutrition, provide health education for mothers, and preschool for children 2 - 6 years old in 1975. Many children attend their local anganwadis, while others attend private schools, and others do not attend school at all. A pre-tested questionnaire was used to interview 125 urban and 130 rural mothers regarding their knowledge, attitudes, and practices about acute diarrheal disease (ADD), acute respiratory infections (ARI), and nutrition (practice only) as they pertained to their 2 - 5 year-old child. Two-week and four-week health recalls were obtained to determine which children had experienced diarrhea or ARIs during those time periods. Anthropometric measurements of the children (weight, height, upper-arm circumference) were collected whenever possible. The study was carried out in an urban slum rural villages surrounding in and surrounding Bangalore, India. Data was collected from March through May of 2009. Through data analysis, KAP and child health scores were calculated to compare four preschool types: anganwadis receiving health check-ups from a medical college, anganwadis not receiving the medical check- ups, other (non-anganwadi) preschools and children not attending preschool. Analyses were performed to identify gaps in KAP, determine the impact of KAP on nutritional status, determine the impact of KAP on ADD and ARI, and determine if preschool type influences KAP scores. Children not attending preschool of any type are at higher risk of ADD, ARI, and being underweight. These children have mothers with the lowest attitude scores. Mothers of children in other preschools have the highest percentage of good knowledge and practice scores. Children who attend other preschools also have the lowest prevalence of underweight. This information can be useful in designing interventions for specific populations.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.