Rapid enzymatic detection of organophosphorous and carbamate pesticides in water

The increased use of pesticides has resulted in a corresponding increase in concern for the effect they may have on the health of humans and other non-target organisms. The two main areas of concern are the toxicological effects that mixtures of pesticides may have as well as the endocrine disrupting effects. Although the individual pesticides may be present at concentrations below the levels deemed to be detrimental to health, it has been argued that their combined effect may still result in elevated health risks. Another important aspect of pesticide risk assessment requires a consideration of the breakdown products of pesticides and their effect on human health. There has been very little research into the effects of degradation products and this issue should be addressed as these could potentially pose a higher risk than their parent compounds. One of the most important bio-markers available for use is the ubiquitous enzyme acetylcholinesterase (AChE). This enzyme is responsible for one of the most important functions in the body; namely nerve impulse transmission, upon which all life depends. The inhibition of this enzyme indicates toxicity and as a subsequence, a threat to the organism’s well-being. Bioassays have also recently been developed to test chemicals for endocrine disrupting effects. These tests rely on a dose response equivalent to that of the most potent well known estrogen 17-β estradiol. Any chemical that has a measurable response is deemed to display endocrine disrupting effects. This first aim of this study was to investigate the toxicological and endocrine disrupting effects of three organophosphorus pesticides; aldicarb, parathion and demeton-S-methyl, in addition to two breakdown products; aminophenol and p-nitrophenol. Two carbamate pesticides; carbaryl and carbofuran were also analysed. The toxicological effects of mixtures of the parent pesticide compounds were tested to assess if any antagonistic, additive or synergistic effects were observed. This data was then used in conjunction with an artificial neural network to assess if individual pesticides could be distinguished from mixtures of pesticides. A final objective was to sample various Eastern Cape water sources, utilising the enzymatic assay to determine the presence of any of these pesticides in these samples. There were several conclusions drawn from this study. AChE was successfully used as an assay to test the toxicity of the pesticides under investigation, based on their inhibition of this enzyme. An important factor for consideration throughout the study was the need to establish basal and monitor AChE activity (i.e. the need to monitor AChE activity in the absence of any pesticide). This ensured accurate comparison of the results obtained. It was found that demeton-S-methyl was the most potent of these pesticides followed by carbaryl, parathion, aldicarb and finally carbofuran, and that carbofuran could potentiate AChE. The results indicated that pesticide mixtures generally exhibited an additive inhibitory effect on AChE, although at some concentrations of pesticides, synergistic and antagonistic effects were noted. From the data using mixtures of pesticides, a feed forward neural network was created that was successfully able to distinguish individual pesticides from mixtures within its training parameters. None of the pesticides tested displayed endocrine disrupting properties in the Yeast Estrogen Screen (YES), T47D-KBluc and MDA-kb2 bio-assays. Other studies reported mixed results in this regard and thus no final conclusions could be drawn. The Blaauwkrantz River, Kariega River, Sundays River, Swartkops River and Kowie River were all tested for pesticides and although positive results were recorded, conventional methods indicated that there were no pesticides in the rivers. There were, however, trace metals present which are known to inhibit AChE, thus causing a false positive result. These results indicated that AChE can be used as a high throughput initial pre-screening tool, but that it cannot serve as a substitute for more accurate conventional testing methods

Contribution of Maternal Immunity to Decreased Rotavirus Vaccine Performance in Low- and Middle-Income Countries

ABSTRACT The role of maternal immunity, received by infants either transplacentally or orally from breast milk, in rotavirus vaccine (RV) performance is evaluated here. Breastfeeding withholding has no effect on vaccine responses, but higher levels of transplacental rotavirus-specific IgG antibody contribute to reduced vaccine seroconversion. The gaps in knowledge on the factors associated with low RV efficacy in low- and middle-income countries (LMIC) remain, and further research is needed to shed more light on these issues

Association of Maternal Immunity with Rotavirus Vaccine Immunogenicity in Zambian Infants

IntroductionLive attenuated oral vaccines against rotavirus (RV) have been shown to be less efficacious in children from developing countries. Reasons for this disparity are not fully understood. We assessed the role of maternal factors including breast milk RV-specific IgA, transplacentally acquired infant serum RV-specific IgG and maternal HIV status in seroconversion among Zambian infants routinely immunized with Rotarix™ (RV1).Methods420 mother-child pairs were recruited at infant age 6–12 weeks in Lusaka. Clinical information and samples were collected at baseline and at one month following the second dose of RV1. Determination of breast milk RV-specific IgA and serum RV-specific IgA and IgG was done using standardized ELISA. Seroconversion was defined as a ≥ 4 fold rise in serum IgA titre from baseline to one-month post RV1 dose 2, while seropositivity of IgA was defined as serum titre ≥ 40 and antibody variables were modelled on log-base 2. Logistic regression was used to identify predictors of the odds of seroconversion.ResultsBaseline infant seropositivity was 25.5% (91/357). The seroconversion frequency was 60.2% (130/216). Infants who were IgA seropositive at baseline were less likely to seroconvert compared to their seronegative counterparts (P = 0.04). There was no evidence of an association between maternal HIV status and seroconversion (P = 0.25). Higher titres of breast milk rotavirus-specific IgA were associated with a lower frequency of seroconverson (Nonparametric test for trend Z = -2.84; P<0.01): a two-fold increase in breast milk RV-specific IgA titres was associated with a 22% lower odds of seroconversion (OR = 0.80; 95% CI = 0.68–0.94; P = 0.01). There was seasonal variation in baseline breast milk rotavirus-specific IgA titres, with significantly higher GMTs during the cold dry months (P = 0.01).ConclusionLow immunogenicity of RV1 vaccine could be explained in part by exposure to high antibody titres in breast milk and early exposure to wild-type rotavirus infections. Potential interference of anti-RV specific IgA in breast milk and pre-vaccination serum RV specific-IgA and IgG titres with RV1 seroconversion and effectiveness requires further research

Effect of innate antiviral glycoproteins in breast milk on seroconversion to rotavirus vaccine (Rotarix) in children in Lusaka, Zambia.

INTRODUCTION: Rotavirus vaccines have been introduced into national immunization programmes to mitigate morbidity and mortality associated rotavirus diarrhoea. Lower vaccine effectiveness has however been noted in low-middle income countries, but little is known about the role of maternal components found in breast milk. This study assessed the effect of lactoferrin, lactadherin, and tenascin-c on rotavirus vaccine seroconversion. METHODS: This was a retrospective cohort study of 128 infants who had been fully immunized with Rotarix™. Serum samples were collected from the infant at baseline and one month after second rotavirus vaccine dose. Breast milk samples were collected from mothers at baseline. Standard ELISA was used to determine titres of rotavirus-specific immunologlobulin G and A in breast milk and serum as well as concentrations of lactoferrin, lactadherin, and tenascin-c. Poisson regression model with robust standard error was used to estimate the effect of breast milk components on seroconversion. The components were modelled on log base 2 so that the effect would be interpreted as a doubling of the concentration. RESULTS: In a multivariable analysis adjusting for maternal age, maternal HIV status, seropositivity at baseline, sex, age of child at vaccination as well as breast milk IgA and IgG, we found evidence of independent effect of LA (Adjusted IRR = 0.95; 95% CI = 0.91-0.99; P = 0.019) on seroconversion while there was no evidence for TNC (Adjusted IRR = 1.00; 95% CI = 0.85-1.17; P = 0.967) and LF (Adjusted RR = 1.01; 95% CI = 0.96-1.05); P = 0.802). We explored the joint effects of the three components but we found no evidence (Adjusted RR = 0.95; 95% CI = 0.81; P = 0.535). CONCLUSION: High breast milk concentrations of lactadherin might play a role in infant's failure to seroconvert to rotavirus vaccines. Further research to understand this observed association is an important consideration

Contribution of Maternal Immunity to Decreased Rotavirus Vaccine Performance in Low- and Middle-Income Countries

The role of maternal immunity, received by infants either transplacentally or orally from breast milk, in rotavirus vaccine (RV) performance is evaluated here. Breastfeeding withholding has no effect on vaccine responses, but higher levels of transplacental rotavirus-specific IgG antibody contribute to reduced vaccine seroconversion. The gaps in knowledge on the factors associated with low RV efficacy in low- and middle-income countries (LMIC) remain, and further research is needed to shed more light on these issues

Early linear growth retardation: results of a prospective study of Zambian infants

Abstract Background Linear growth retardation is the most dominant nutritional problem globally. We aimed to describe linear growth trajectory among infants under 2 years of age using the WHO growth velocity standards. Method This was a prospective cohort study of infants enrolled at 6 weeks of age and followed up for up to 24 months in Kamwala Urban Health Centre, Lusaka, Zambia. The study was conducted between April 2013 and March 2015. Infants were enrolled if they were 6–12 weeks of age and the mother was willing to participate voluntarily and provided informed consent. Anthropometric data were collected at scheduled clinic visits at 1 month, 2 months, 3 months, then quarterly until the infant was 24 months old. We defined linear growth velocity as the rate of change in height. We estimated linear growth velocity as the first derivative of the penalized cubic spline mixed effects model. Results A total of 338 children were included in the analysis. Of these, 185 (54.7%) were female, 115 (34.1%) were born to HIV positive mothers and thus classified as HIV Exposed (HE). The mean age of children at enrollment was 1.6 months (SD = 0.15). On average, the growth velocity for 3-month length increments conditional on age were 0–3 months = 2.97 cm/3mo (95%CI = 2.69, 3.25); 3–6 months = 2.62 cm/3mo (95%CI = 2.38, 2.87); 6–9 months = 1.57 cm/3mo (95%CI = 1.43, 1.71); 9–12 months = 1.18 cm/3mo (95%CI = 1.08, 1.28); 12–15 month = 1.14 cm/3mo (95%CI = 1.02, 1.27); 15–18 months = 0.87 cm/3mo (95%CI = 0.79, 0.96); 18–21 months = 0.80 cm/3mo (95%CI = 0.72, 0.89); and 21–24 months = 0.86 cm/3mo (95%CI = 0.77, 0.96). For both boys and girls, the growth velocity in our cohort were consistently below the 3rd percentile of the WHO linear growth velocity standard. The estimated mean height and the age at which growth begins to falter were 68.6 cm (95%CI = 68.0, 69.2) and 13.6 months (95%CI = 13.2, 14.1) respectively. Conclusion We found slower rate of growth among otherwise healthy Zambian infants. The data suggests that growth retardation is universal and profound in this cohort and may have already been occurring in utero

Immunogenicity of rotavirus vaccine (RotarixTM) in infants with environmental enteric dysfunction.

Deployment of rotavirus vaccines has contributed to significant declines in diarrheal morbidity and mortality globally. Unfortunately, vaccine performance in low-middle income countries (LMICs) is generally lower than in developed countries. The cause for this has been associated with several host and maternal factors including poor water sanitation and hygiene (WASH) status, which are predominant in LMICs. More recently, environmental enteric dysfunction (EED) has specifically been hypothesized to contribute to poor vaccine uptake and response. The aim of this study was to examine the association between serological biomarkers of EED and seroconversion to rotavirus vaccine in Zambian infants.This was a retrospective cohort study of 142 infants who had been fully immunized with Rotarix™, and had known seroconversion status. Seroconversion was defined as 4-fold or more increase in rotavirus-specific IgA titres between pre-vaccination and one month post-dose two vaccination. We performed ELISA assays to assess soluble CD14 (sCD14), Endotoxin Core IgG Antibodies (EndoCAb), intestinal fatty acid binding protein (i-FABP) and Zonulin according to the manufacturers protocols. Generalised linear model with family-poisson, link-log and robust standard error was used to estimate the independent effects of biomarkers on seroconversion adjusting for important cofounders.The median concentration of Zonulin, Soluble CD14, EndoCaB, and IFABP were 209.3 (IQR = 39.7, 395.1), 21.5 (IQR = 21.5, 21.5), 0.3 (IQR = 0.3, 0.3), and 107.7 (IQR = 6.4, 1141.4) respectively. In multivariable analyses adjusting for the independent effect of other biomarkers and confounders (i.e. age of child at vaccination, breast-milk anti-rotavirus IgA, infant serum anti-rotavirus IgG, and IgA seropositivity at baseline), there was strong evidence of about 24% increase in seroconversion due to doubling Zonulin concentration (Adjusted risk ratio (aRR) = 1.24; 95% CI = 1.12 to1.37; p<0.0001). Similarly, we found about 7% increase in seroconversion due to doubling IFABP concentration (aRR = 1.07; 95% CI = 1.02 to 1.13; p = 0.006).We found that high levels of zonulin and IFABP played a role in seroconversion. It is plausible that increased gut permeability in EED allows greater uptake of the live virus within the vaccine, but later consequences result in deleterious local structural distortions and malabsorption syndromes

In-vitro inhibitory effect of maternal breastmilk components on rotavirus vaccine replication and association with infant seroconversion to live oral rotavirus vaccine.

BackgroundDespite contributing to a significant reduction in rotavirus associated diarrhoea in highly burdened low- and middle-income countries, live attenuated, oral rotavirus vaccines have lower immunogenicity and efficacy in these settings in comparison to more developed countries. Breastmilk has been implicated among factors contributing to this lowered oral vaccine efficacy. We conducted in-vitro experiments to investigate the inhibitory effects of maternal antibody and other non-antibody components in breastmilk on rotavirus vaccine strain (Rotarix) multiplication in MA104 cell culture system and assessed associations with in-vivo vaccine seroconversion in vaccinated infants.MethodsBreastmilk samples were collected from mothers before routine rotavirus vaccination of their infant at 6 weeks of age. For each sample, whole breastmilk, purified IgA, purified IgG and IgG and IgA depleted breastmilk samples were prepared as exposure preparations. A 96 well microtitre plate was set up for each sample including a control in which only MA104 cells were grown as well as a virus control with MA104 cells and virus only. The outcome of interest was 50% inhibition dilution of each of the exposure preparations calculated as the titer at which 50% of virus dilution was achieved. Samples from 30 women were tested and correlated to vaccine seroconversion status of the infant. HIV status was also correlated to antiviral breastmilk proteins.ResultsThe mean 50% inhibitory dilution titer when whole breastmilk was added to virus infected MA104 cells was 14.3 (95% CI: 7.1, 22.7). Incubation with purified IgG resulted in a mean 50% inhibitory dilution of 5 (95%CI -1.6, 11.6). Incubating with purified IgA resulted in a mean 50% inhibitory dilution of 6.5 (95% CI -0.7, 13.7) and IgG and IgA depleted breastmilk did not yield any inhibition with a titer of 1.06 (95%CI 0.9, 1.2). Higher milk IgA levels contributed to a failure of infants to seroconvert. HIV was also not associated with any antiviral breastmilk proteins.Discussion and conclusionWhole breastmilk and breastmilk purified IgG and IgA fractions showed inhibitory activity against the rotavirus vaccine Rotarix™ whilst IgA and IgG depleted breastmilk with non-antibody breastmilk fraction failed to show any inhibition activity in-vitro. These findings suggest that IgA and IgG may have functional inhibitory properties and indicates a possible mechanism of how mothers in rotavirus endemic areas with high titres of IgA and IgG may inhibit viral multiplication in the infant gut and would potentially contribute to the failure of their infants to serocovert. There was not association of HIV with either lactoferrin, lactadherin or tenascin-C concentrations

Association of biomarkers of enteric dysfunction, systemic inflammation, and growth hormone resistance with seroconversion to oral rotavirus vaccine: A lasso for inference approach.

BackgroundRotavirus gastroenteritis remains a leading cause of morbidity and mortality despite the introduction of vaccines. Research shows there are several factors contributing to the reduced efficacy of rotavirus vaccines in low- and middle-income settings. Proposed factors include environmental enteric dysfunction (EED), malnutrition, and immune dysfunction. This study aimed to assess the effect of these factors on vaccine responses using a machine learning lasso approach.MethodsSerum samples from two rotavirus clinical trials (CVIA 066 n = 99 and CVIA 061 n = 124) were assessed for 11 analytes using the novel Micronutrient and EED Assessment Tool (MEEDAT) multiplex ELISA. Immune responses to oral rotavirus vaccines (Rotarix, Rotavac, and Rotavac 5D) as well as a parenteral rotavirus vaccine (trivalent P2-VP8) were also measured and machine learning using the lasso approach was then applied to investigate any associations between immune responses and environmental enteric dysfunction, systemic inflammation, and growth hormone resistance biomarkers.ResultsBoth oral and parenteral rotavirus vaccine responses were negatively associated with retinol binding protein 4 (RBP4), albeit only weakly for oral vaccines. The parenteral vaccine responses were positively associated with thyroglobulin (Tg) and histidine-rich protein 2 (HRP2) for all three serotypes (P8, P6 and P4), whilst intestinal fatty acid binding protein (I-FABP) was negatively associated with P6 and P4, but not P8, and soluble transferrin receptor (sTfR) was positively associated with P6 only.ConclusionMEEDAT successfully measured biomarkers of growth, systemic inflammation, and EED in infants undergoing vaccination, with RBP4 being the only analyte associated with both oral and parenteral rotavirus vaccine responses. Tg and HRP2 were associated with responses to all three serotypes in the parenteral vaccine, while I-FABP and sTfR results indicated possible strain specific immune responses to parenteral immunization

Cumulative frequency profiles of RV-IgA titres in pre dose 1 breastmilk of 216 mother-infant pairs by infant IgA seroconversion post dose 2 of rotavirus vaccine.

<p>Seroconversion was defined as four-fold increase in rotavirus-specific IgA titres in post dose 2 sera when compared to the corresponding pre-immunization sera.</p