Ensayos realizados

Although this research has filled some of the gaps in knowledge relating to breast milk immunology and Cryptosporidium-risk factors in young infants, there were some limitations. My sample size was relatively small and may not have been powerful enough to detect differences in some of my outcome variables. This study was a pilot

study intended to generate data for future, larger studies and the sample size was calculated to detect differences in TNFα in maternal serum of HIV-positive and HIV- negative women. Indeed, I was able to detect significant differences in maternal serum TNFα based on maternal HIV-status. Future studies should aim to recruit a larger sample size, especially given the difficulty in collecting infant serum samples, which was a limiting factor in my study. Based on the results of my study, I have calculated the sample size required to detect differences in breast milk immune molecules by maternal HIV-status; results are presented in Table 5.1. According to these sample size

calculations, the current study was underpowered to find significant differences in breast milk immune molecules between HIV-positive and HIV-negative women, especially with regards to the immunoglobulins.

Table 5.1 Sample size requirements for future studies

Immune Molecule HIV+ Mean HIV+ SD HIV- Mean HIV- SD n/group*

IgA 220.3 186.5 223.7 224.6 57867 IgG1 1016.5 1628.0 765.3 987.6 451 IgG2 3857.4 7217.2 3683.6 5963.3 22775 IgG3 23.7 32.6 21.4 23.6 2404 IgG4 13.6 27.3 12.9 20.4 18605 IgM 332.0 250.5 372.9 371.6 943 IFNγ 2.29 0.18 7.95 39.02 374 IL-6 5.09 6.54 5.83 16.83 4673 IL-12 2.32 0.30 2.50 1.02 274 IL-13 2.45 0.64 2.34 0.42 381 IL-15 24.62 26.16 19.57 15.6 286 TNFα 7.13 8.33 6.38 8.42 1958 * α = 0.05; power = 0.8

In both Chapter 2 and Chapter 4, I noted that water-related exposures were a risk for infant Cryptosporidium infection; in Chapter 2, maternal hand washing was

associated with an increased risk of infant Cryptosporidium infection, while in Chapter 4, both water source and the consumption of unboiled water were associated with infant Cryptosporidium. It is important for future studies to not only analyze fecal samples, but also water samples from the source and from the home in order to help determine

transmission pathways. My study identified Cryptosporidium infection using

microscopy, which is unable to differentiate between different species of the pathogen. Studies show that 90% of human Cryptosporidium infections are due to just two species,

namely C. parvum and C. hominis [15]. Previous research has indicated that there may be difference in the epidemiology and clinical implications based on the infective species of Cryptosporidium. It appears that the main reservoir for C. parvum infection is

zoonotic, while C. hominis appears to infect humans exclusively [15]. In addition to water testing, genotyping of the Cryptosporidium species would help shed light on the mode of transmission thus allowing better planning and implementation of interventions to prevent Cryptosporidium infection.

Many studies have linked low CD4 cell counts of HIV-positive individuals with a higher incidence and severity of Cryptosporidium [20], yet none of these studies have considered CD4 cell counts of HIV-negative individuals. In order to get a more complete picture of the way in which CD4 cell counts are related to Cryptosporidium risk, future studies should collect CD4 cell counts for all participants, not just HIV-positive

individuals.

The intrauterine environment is the first exposure the infant has to the maternal environment and to the maternal immune system. Many immune molecules are transferred to the infant transplacentally and via cord blood [29-31], thus maternal Cryptosporidium infection during pregnancy may impact an infant’s risk of future infection. Collecting and analyzing maternal fecal samples during pregnancy may help determine if intrauterine exposure to maternal Cryptosporidium infection is associated with Cryptosporidium infection during infancy. Additionally, since infant

Cryptosporidium prevalence rose dramatically between Month 3 and Month 6, the collection of fecal samples at monthly intervals (Month 4 and Month 5) between these visits would help our understanding of the natural history of infection in infants. During

the period between Month 3 and Month 6, infants often undergo dramatic changes in feeding and therefore this period is especially crucial in understanding the causal relationship between infant feeding practices and Cryptosporidium infection.

Another limitation of my study was that we only analyzed total concentrations of breast milk immunoglobulins rather than antigen-specific immunoglobulins. Breast milk IgA in the infant intestine acts to exclude antigen from circulation by preventing

penetration through the gut epithelium [32]. Based on the immunology of the entero- mammary system, antigen encountered in the maternal intestine stimulates the production of antigen-specific IgA, which is then secreted in breast milk. A previous study

determined that anti-Cryptosporidium IgA targeted the surface of C. parvum oocysts leading to the conclusion that Cryptosporidium-specific IgA in breast milk is capable of protecting the breastfeeding infant from infection [10]. Indeed, the same study showed that infants consuming higher concentrations of Cryptosporidium-specific IgA in breast milk were at decreased risk of becoming infected [10]. Future research should focus on Cryptosporidium-specific IgA in breast milk rather than simply total breast milk IgA in order to better determine how breast milk immunoglobulins are related to protection from Cryptosporidium infection.

Overall, the results of this prospective cohort study demonstrate that

Cryptosporidium is an under-recognized public health problem for lactating mothers and young infants in rural Tanzania. These results indicate that infant and young child feeding practices are associated with early Cryptosporidium infection, and that feeding practices remain sub-optimal. Public health programs should focus on education and support for exclusive breastfeeding and appropriate complementary feeding during the

antenatal and post-partum periods. Findings of this study also inform future research priorities, namely the testing of household water samples, analysis of Cryptosporidium- specific breast milk antibodies, and genotyping of infective Cryptosporidium species in order to further refine our understanding of the epidemiology of Cryptosporidium infection and transmission.

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APPENDIX A