Immunological effects of asymptomatic vibrio cholerae, salmonella enterica serovar typhi and entamoeba histolytica enteric pathogens during HIV infection and exposure.
Nhidza, Agness Farai.
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Background Asymptomatic infection by enteric pathogens has been attributed to causing several outbreaks in the world. Common pathogens in this category are Vibrio cholerae, Salmonella typhi and Entamoeba histolytica infection of pregnant and lactating mothers. Exposure of the infant and the immune response during the early developmental years requires investigation. In the era of high HIV burden and the introduction of HIV prevention of mother to child transmission (PMTCT), many infants are exposed to these pathogens yet remain uninfected, however still face the challenge of high morbidity and mortality as compared to their unexposed and uninfected counterparts. The cause of such an anomaly has not been fully investigated. Aim The aim of this study is to investigate the burden and the immunological effect of asymptomatic enteric pathogen carriage in pregnant mothers, both HIV-infected and HIV-uninfected, and their infants in a high HIV burdened population of Harare, Zimbabwe. Methodology The study was a purposive cross-sectional sub-study in a birth cohort established at the University of Zimbabwe, College Of Health Sciences (UZ-CHS Birth Cohort). Participants were from the highdensity suburbs of Glenview, Kuwadzana and Dzivarasekwa. These suburbs were previously affected by the 2008 cholera epidemic as well as the 2012 typhoid outbreak that hit Harare, Zimbabwe. HIV rapid testing was performed at enrolment for mothers unaware of their HIV status or those who wanted HIV re-testing for various personal reasons. Those HIV infected with documented HIV status were asked to provide the evidence and were not re-tested. The HIV status of neonates was ascertained using nucleic acid-based testing at the National Early Infant Diagnostic (EID) department based at the National Microbiology Reference Laboratory in Harare, Zimbabwe. The test made use of the Dried Blood Spot (DBS) samples collected from the infants at various time points. A total of 417 pregnant mothers were recruited based on their consent to provide stool samples, blood and urine for both the mother at recruitment and the infant upon delivery. A questionnaire to collect information on socio-demographic, clinical, delivery, water and sanitation, diarrheal history including antibiotic use, concurrent TB infections, ART regimen, planned baby feeding method, diet as well as nutritional data on probiotic uptake was administered to the mothers. Of the 417 mothers, only 204 mothers and 308 infants provided stool samples, which were cultured for enteric pathogens targeting Salmonella entrica serovar Typhi (S. typhi) and Vibrio cholerae, although in the process any non-lactose fermenter identified was considered a potential pathogen hence were further identified using an Analytical Profile Index (API; Biomerieux: France) and included in the study. The stool samples were also used for antigen ELISA targeting Entamoeba histolytica (E. histolytica) infection. The microbial results were used to calculate the prevalence of S. typhi, V. cholerae and E. histolytica in the study population. Data was analysed using STATA 13 analytical software. For further analysis on immunological factors in mother-infant pairs (MIPs), only paired mothers were included based on stool as well as plasma availability and the outcome of culture and ELISA results. Multiplex immunoassay utilised a 27-plex (IL-1β, IL-1r, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL12p70, IL-13, IL-15, IL-17a, Eotaxin, basic PDGF, G-CSF, GM-CSF, IFN-, IP-10, MCP1(MCAF), MIP-1α, MIP-1β , PDGF-BB, RANTES, TNF- α and VEGF) cytokine profile and a 6- plex isotyping (immunoglobulin: IgG1, IgG2, IgG3, IgG4, IgM and IgA) assay. Immunological assays were performed on the 39 MIPs purposively selected based on the culture outcomes as described, using BioRad Luminex machine according to manufacturer’s instruction Based on the cytokine and immunoglobulin concentrations, maternal characteristics influencing infant cytokine and immunoglobulin production were analysed. Results The presence of V. cholerae nor S. typhi was detected but E. histolytica prevalence in mothers was calculated at 5.4% while in infants it was 5.6%. There was no significant difference in the prevalence of E. histolytica between HIV-infected and HIV-uninfected mothers (p=0.318) as well as between HIV-exposed uninfected and HIV-unexposed uninfected infants (p=0.056). E. histolytica infection was only noted in 1 HIV-uninfected mother-neonate pair. The resistance to 3rd generation cephalosporines in the non-lactose fermenting bacterial isolates was detected. The cytokine groups of IL-1r, IL-4, IL-9, IL-12p70, IL-17a, G-CSF and PDGF-BB were significantly raised in E. histolytica infected compared to non-infected lactating mothers (p<0.05). In babies, E. histolytica carriage had no significant effect on the cytokine and immunoglobulin concentration. In this study maternal E. histolytica carriage was significantly (p=0.026) correlated with infant Interleukin-12p70 (IL-12p70; p<0.026), Fibroblast Growth Factor-basic (FGF-basic; p<0.026), Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF; p<0.026) and Tumor Necrosis Factor (TNF; p<0.026)-α. The odds ratio of passing high levels of these cytokines to infants is reduced by 86% if the mother is a carrier of E. histolytica during pregnancy (OR: 0.14). Carriage of any form of enteric infection such as Non-lactose fermenters (NLFs) including E. histolytica significantly increased the levels of IL-1r, IL-4, IL-9, IL-10, IL-12p70, IL17a, G-CSF, GM-CSF, IFN-, PDGF-BB and TNF-α cytokines (p<0.05). However, no significant difference in immunoglobulin levels was noted amongst the mothers. IL13 and G-CSF levels were significantly raised in HIV-infected compared to HIV-uninfected lactating mothers (p=0.0389 and p=0.0324, respectively). IgA immunoglobulin levels were significantly raised among HIV-exposed uninfected than HIVunexposed uninfected infants. It has also been observed that maternal E. histolytica carriers are significantly more likely to have infants with low levels of IL-12p70, FGF-basic, GM-CSF and TNF-α cytokines and high levels of IgA immunoglobulin. HIV-infected mothers are significantly more likely to have infants with low levels of IgG2 and IgA immunoglobulins. Mothers giving birth at a mean age above 30.38 ±STD 6.09 years are more likely to have infants with low IgG4 levels (p=0.050). Maternal HIV infection influences infant IgG2 and IgA immunoglobulin levels. Also, a maternal age of >30years reduces the odds ratio of passing high IgG4 to the infant (p=0.050). Maternal HIV infection influences infant IgG2 and IgA immunoglobulin levels. Infants born to HIV positive mothers are significantly associated (p=0.049) with lower odds of having high IgG2 levels, ie., reduced by 76% in infants born to HIV-infected mothers compared to those born to HIV-uninfected mothers. Similarly, an infant born to a HIVinfected mother is significantly associated (p=0.035) with lower odds of having high IgA immunoglobulin levels compared to those born to HIV-uninfected mothers (OR: 0.22). Underweight/small to gestational age (SGA) babies (birth weight <2500g) were found to have significantly raised IL-7 cytokine levels compared to normal for gestational age (NGA) babies whose birth weight was >2500g (p=0.0149). Conclusion Asymptomatic enteric E. histolytica carriage is a reality in pregnant women and their neonates with the possibility of congenital transmission. We confirm that there is no V. cholerae carriage in Glenview, Dzivarasekwa and Kuwadzana, areas affected in the 2008 cholera epidemic in Zimbabwe. Moreover, there was no evidence of S. typhi carriage in these suburbs following the devastating 2012 typhoid outbreak. This study reports an expansive pro-inflammatory cytokine and chemokine profile that is up-regulated in lactating mothers with asymptomatic enteric pathogens. HIV exposure had no effect on baby cytokine profiles. The importance of IL-7 in neonates requires further investigation as it might assist in reducing the prevalence of small for gestational age (SGA) babies. Significance and impact of study Our study culminates in the need to consider maternal characteristics such as age, HIV status and enteric pathogen carriage in pregnancy as these might impact on the infants’ immune responses. This will assist in the decisions on disease treatment and management of pregnant women and infants. The study findings also assist in developing effective immunotherapy in E. histolytica and HIV exposures and infections in an effort to promote maternal and child health.