Association of Maternal Toxoplasma Gondii Molecular and Serological Positivity With Child's Gross-Motor Development and Behavior in Tribal Regions of Gujarat, India: A Prospective Study Aarthi Sundararajan, Kranti Vora, Shahin Saiyed, Senthilkumar Natesan, Vidhi Vaidya Health Science Reports, 2024 Toxoplasma gondii is a protozoan parasite causing toxoplasmosis in humans, with lifelong presence in brain and muscular tissues [1, 2]. Disease is mostly asymptomatic with infection largely correlating with changes in human personality [3, 4]. T. gondii infection can be acquired from environment and during pregnancy when the parasite can be transmitted to the fetus through the placenta resulting in congenital toxoplasmosis. National and international studies including our previous community study have assessed Toxoplasma prevalence in pregnant women through molecular and serological methods [5-8]. Follow-up studies have explored severe or more apparent outcomes among children born to Toxoplasma-positive mothers, such as hearing-vision deficiencies including chorioretinitis [9, 10]. Longer pregnancy, slower fetal development, and slower post-natal gross-motor development have been associated with children born to Toxoplasma-positive mothers [11, 12]. However, similar profiling of development characteristics in a developing country like India is lacking. In addition, temperament characteristics among children born to Toxoplasma-positive mothers are unclear. Therefore, the objective of this small-scale, exploratory study was to explore the association of maternal Toxoplasma positivity with child's gross-motor development and temperament through structured, well-established, parent-reported questionnaires. This is a nested investigation involving mothers from a previously completed prospective pregnancy cohort study [6]. T. gondii prevalence study was previously conducted among pregnant women in Modasa-Bhiloda blocks, Aravalli district, Gujarat, India covering > 100 villages through universal sampling [6, 13]. A combination of nested PCR assays detecting B1 gene and ELISA assays detecting Toxoplasma-specific IgM and IgG antibodies were employed. The study revealed high seroprevalence (76.5%) owing to poor personal and environmental hygiene in these tribal regions [6, 13] providing relevance for conducting the present follow-up study. Following categories of Toxoplasma positivity in mothers were observed: PCR−IgG+ (40.9%), PCR+IgG+ (34.5%), PCR+IgG− (3.1%), and PCR+IgG+IgM+ (1.1%). Based on this data set of mothers, the target population for the present study included only the children born to the two highest prevalence categories of Toxoplasma positivity (PCR+IgG+ and PCR−IgG+). Villages demonstrating the highest prevalence in both categories of Toxoplasma positivity were included in the present study. For optimal use of resources, Toxoplasma-positive mothers in the surrounding villages were also included due to geographical proximity for easy movement of data collectors between villages. Within these selected villages, all women who participated in the previous prevalence study were contacted, demonstrating the three categories of Toxoplasma status (PCR+IgG+, PCR−IgG+, PCR−IgG−) without adverse birth outcomes. However, not all women in each village were available due to migration. Children born to available Toxoplasma-positive (PCR+IgG+ [n = 95] and PCR−IgG+ [n = 94]) and Toxoplasma-negative (PCR−IgG− [n = 59]) mothers were enrolled. The sample size constituted 30% of the pregnancy cohort study population demonstrating these categories of Toxoplasma status without adverse birth outcomes. Enrolled children included infants and toddlers between the ages of 6 and 28 months old. Based on the prevalent modes of environmental transmission mechanisms, a questionnaire examining household interaction with cats, milk–water source, and food consumption was administered to parents. Children's bio-parameters including age, height, weight, and gross-developmental milestones were noted. Children's height and weight were measured by the data collector with the help of a measuring tape and weighing scale, respectively. Infant behavior questionnaire- revised, very short version (IBQ-R-VS) was administered to parents, measuring infant's (≤ 12 months old) three scales of temperament; surgency, effortful control, and negative affect. The questionnaire and scoring guidelines were obtained from Dr. Sam Putnam, Bowdoin College, who originally developed the questionnaire [14]. The questionnaire was translated into the local language (Gujarati), back-translated, and pretested in a pilot study. Cronbach's alpha coefficient was determined for scales; surgency, negative affect, and effortful control as 0.56, 0.66, and 0.55, respectively. All questionnaires were administered to parents and responses were recorded by a trained data collector. Two-sided significance was determined for all statistical tests and p ≤ 0.05 was considered as statistically significant. Shapiro–Wilk test of normality was performed to determine the nature of distribution among the groups being compared. All statistical analyses were performed using the Statistical Package of Social Science (IBM SPSS Statistics 26). The study was approved by the Institutional Ethics Committee of the Indian Institute of Public Health Gandhinagar (approval code: 15/2020-21). The study implications were explained to the parents of study participants in the local language (Gujarati). Written informed consent was obtained from parents of study participants in Gujarati. For illiterate parents, a thumbprint was taken along with the witness's signature. No invasive procedures or interventions were performed. There were no significant differences in the prevalence of any of the household-associated infection risk factors among the categories of children born to Toxoplasma-positive mothers (IgG+, PCR+IgG+, PCR−IgG+) and Toxoplasma-negative mothers (PCR−IgG−) (Supporting Information: Table S1). Comparing children born to Toxoplasma-positive and Toxoplasma-negative groups, there were no significant differences in bio-parameters such as age, low birth weight status, weight, and height (Table 1). Adjustment for confounders such as gender, low birth weight status, and age did not result in any significant association of mothers' Toxoplasma positivity with the child's weight or height. In contrast to another study [12], gross motor skill development did not show significant differences between categories of children born to Toxoplasma-positive and Toxoplasma-negative mothers (Table 1). Ages of achieving indicated milestones are within the age cut-offs for normal development as per the Indian Academy of Pediatrics Guidelines [15], except for the median age of holding head which is delayed by a month from the cut-off in all the groups. None of the children had vision and hearing deficiencies. IBQ-R-VS responses collected from infants (≤ 12 months old) revealed significantly higher levels of negative affect scores in infants born to PCR+IgG+ mothers compared to both infants born to PCR−IgG− mothers, p = 0.01 and infants born to PCR−IgG+ mothers, p = 0.02 (Table 2). No significant changes were observed in other temperament scales. Our study has identified similar exposure to infection risk factors between the households of Toxoplasma-positive (PCR+IgG+, PCR−IgG+, and IgG+) and Toxoplasma-negative mothers (PCR−IgG−). Given this scenario, our study demonstrates that the higher negative affect scores are associated with infants born to PCR+IgG+ mothers, not with infants born to PCR−IgG+ mothers. Although the biological significance of these subsets is presently unknown, our study indicates the importance of performing both PCR and serological assays for better understanding of the relationship of maternal Toxoplasma positivity during pregnancy with a child's temperament characteristics. Large-scale studies are needed to confirm these findings. In addition, longitudinal studies are needed to assess the temperament of these children as they enter adolescence and adulthood. High negative affectivity has been identified in children with attention-deficit/hyperactivity disorder (ADHD), a developmental and behavioral disorder [16, 17]. Interestingly, increased levels of severe form of ADHD were correlated with Toxoplasma seropositivity in children [18, 19]. Although we did not clinically evaluate ADHD in this study, future studies will need to explore the predisposition to development of ADHD in children demonstrating both high negative affectivity and Toxoplasma seropositivity to understand the role of Toxoplasma in regulating behavioral functions. Additionally, biological and environmental factors contribute to negative affectivity, including parenting behaviors, household chaos, and maternal emotion expressivity [20], which were not evaluated in the present study. There was a lack of significant difference in children's bio-parameters between the groups. Although large-scale studies are needed to confirm, inherent similarities including food habits, malnutrition, and anemia [21] need to be considered. In contrast to a Western study [12], our analysis did not show a significant difference in gross-motor skills such as the age at which the child learned to lift the head and move/turn around independently. It is possible that there are genetic and environmental factors that need to be considered [22]. A limitation of the study is that developmental features were not assessed comprehensively, for example, through questionnaires like “Ages and Stages” (ASQ), and Developmental Assessment Scale for Indian Infants (DASII) which have been established to detect developmental delay in Indian children [23]. There are other limitations in the study that children's Toxoplasma status could not be determined due to resource and logistical constraints, and could not be subsequently correlated with their temperament and corresponding mother's Toxoplasma status. Analyzing these aspects in future studies would provide more insight into the pathophysiological link between a mother's Toxoplasma status during pregnancy and subsequent child's behavior. Potential confounding factors such as parent's educational levels, family's emotional environment, and children's health factors were not evaluated. Another limitation is that the Cronbach's alpha values in the IBQ-R-VS questionnaire were not excellent in our study and future studies need to assess how the scale reliability in Gujarati-translated questionnaires could be further improved. It is possible that there are other cross-cultural nuances that need to be considered. One of the strengths of this study is that this is a follow-up of a previously conducted large prospective study that assessed the prevalence of TORCH positivity among pregnant women. In the present study, as their children were being evaluated, mothers were enthusiastic in participating and forthcoming in their responses. Furthermore, the participating mothers were not aware of their Toxoplasma status, and therefore no inherent bias was involved while answering the questionnaires. Questionnaires were administered through a data collector belonging to the same study area who ensured that the parents understood the questions. In addition, infection risk factors were not significantly different between the Toxoplasma-positive and -negative mothers, allowing independent interpretation of the child's development and behavioral characteristics. The study indicates the importance of combinatorial assessment of molecular and serological testing for understanding the implications of Toxoplasma positivity during pregnancy through association with the infant's temperament characteristics. Large-scale studies are needed to determine if the PCR+IgG+ category of maternal Toxoplasma status poses an early increased risk for children to develop altered behavior later in life. Such a questionnaire-based approach involving the parents could directly impact understanding of Toxoplasma infection among the tribal community where currently, there is a lack of awareness about the disease. Aarthi Sundararajan: conceptualization, methodology, funding acquisition, project administration, writing–original draft, writing–review and editing, supervision, data curation. Kranti Vora: conceptualization, supervision, project administration, writing–review and editing, methodology. Shahin Saiyed: methodology, project administration, writing–review and editing, data curation. Senthilkumar Natesan: conceptualization, writing–review and editing, supervision. Vidhi Vaidya: writing–review and editing, methodology. The research was funded by the Royal Society of Tropical Medicine and Hygiene in partnership with the National Institute for Health Research as a Small Grant awarded to A.S. (2020). The funder has no inputs in design, implementation, and analysis. The corresponding authors Aarthi Sundararajan and Kranti Vora affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. The study was approved by the Institutional Ethics Committee of the Indian Institute of Public Health Gandhinagar (approval code: 15/2020-21). The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Comparative Profiling of Salivary Cortisol and Salivary DHEA-S among Healthy Pregnant and Non-Pregnant Women Aarthi Sundararajan, Kranti Vora, Shahin Saiyed, Senthilkumar Natesan Hormone and Metabolic Research, 2021 During pregnancy, circulatory cortisol levels increase, remaining steady over the second-third trimester. In contrast, profile of salivary cortisol during pregnancy is debatable, more influenced by factors like time of sample collection in the day. Circulatory DHEA-S decrease by at least 50% over the second-third trimester of pregnancy. However, profile of salivary DHEA-S is unclear. Objective was to determine changes in salivary cortisol and DHEA-S in healthy pregnant women, compared to non-pregnant women during late morning-early afternoon sampling to avoid fluctuations associated with other times. Pregnant women in their second-third trimester prospectively (n=500) and non-pregnant women (n=133) were enrolled in study with informed consent. Live birth outcome with no pregnancy complications and≥2.5 Kg infant birth weight were included. Concentrations of salivary cortisol and DHEA-S were determined through ELISA assays. Compared to non-pregnant women, pregnant women demonstrated significant increases in salivary cortisol [median (interquartile range)=4.2 (5.1) nmol/l vs. 17.2 (13.9) nmol/l, p<0.001] and salivary DHEA-S median (interquartile range)=2.7 (2.9) nmol/l vs. 3.8 (3.2) nmol/l, p<0.001). Consistently, quartile scores representing higher levels of salivary cortisol and DHEA-S concentrations demonstrated significant association with pregnancy. Quartile scores representing higher salivary cortisol/DHEA-S ratio demonstrated significant association with pregnancy. Study suggests the indicated time range of saliva sampling might best parallel the established profile of circulatory cortisol in pregnant women. However, unlike cortisol, study indicates that the salivary DHEA-S profile is distinct from the well-known profile of circulatory DHEA-S during pregnancy. A combinatorial approach involving both salivary and circulatory compartments could provide comprehensive picture of DHEA-S and hypothalamus-pituitary-adrenal axis during pregnancy.
Ramping up of SARS CoV-2 testing for the diagnosis of COVID-19 to better manage the next phase of pandemic and reduce the mortality in India Senthilkumar Natesan, Ragini Bhatia, Aarthi Sundararajan, Kuldeep Dhama, Yashpal S. Malik, Kranti Vora Virusdisease, 2020 The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus-2, a new member of the Coronavirus family. The virus was first identified in Wuhan, China, where the epidemic originated. The viral genome was sequenced and a real time reverse transcription polymerase chain reaction assay was developed and used for the detection of virus. Different countries took different approaches for the diagnosis of COVID-19. Some countries prioritized extensive testing for COVID-19 at a very early phase of the pandemic whereas other countries took a long time to build the testing capacity and to implement the testing extensively. The assay design formats were available in the public domain and thereby allowing researchers to replicate them to make diagnostic kits. Consequently, several antigen or antibody-based diagnostic tests were also developed for the diagnosis of COVID-19. However, there were some validation and regulatory challenges while bringing these assays into the market. During the course of the pandemic, it became clear that the countries which implemented testing at an early stage of the pandemic were capable of controlling the spread more effectively than those that implemented them at later stages. As several countries implemented a lockdown for controlling the spread of the virus, it is critical to build the testing capability to meet the extensive need of testing while exiting the lockdown. Testing and isolation of positive cases are the most effective ways of preventing the spread of virus and gradually returning life back to normality.
Impact of COVID-19 on women and children and the need for a gendered approach in vaccine development Kranti Suresh Vora, Aarthi Sundararajan, Shahin Saiyed, Kuldeep Dhama, Senthilkumar Natesan Human Vaccines and Immunotherapeutics, 2020 The COVID-19 pandemic has imposed unprecedented health and socioeconomic challenges on public health, disrupting it on a global scale. Given that women and children are widely considered the most vulnerable in the times of emergency, whether in war or during a pandemic, the current pandemic has also severely disrupted access to reproductive and child health services. Despite this, data on the effect of the pandemic on pregnant women and newborns remain scarce, and gender-disaggregated indicators of mortality and morbidity are not available. In this context, we suggest the implementation of a gendered approach to ensure the specific needs of women and their newborns are considered during the development of COVID-19 vaccines. Taking into account gender-based biological differences, the inclusion of pregnant and lactating mothers in clinical trials for the development of COVID-19 vaccines is of vital importance.
Methodology considerations for the molecular detection of torch organisms using pcr approaches Bhumika Prajapati, , Kranti Vora, Aarthi Sundararajan, Shahin Saiyed, Ruchi Tiwari, Kuldeep Dhama, Senthilkumar Natesan, , , , , , and Journal of Experimental Biology and Agricultural Sciences, 2020 There is a need for a reliable and cost-effective molecular diagnostic assay for the diagnosis of TORCH [Toxoplasma gondii, Other (Varicella-Zoster virus and Parvovirus B19), Rubella virus, Cytomegalovirus, and Herpes Simplex virus] infections. This would enable early and precise detection of pathogens even in a very low copy number. However, the selection of genomic target, specimen matrix, and different PCR methods can significantly affect the sensitivity and specificity of TORCH molecular detection. This review aimed to provide a comparative analysis of clinical sample types, target nucleotide sequences, and PCR detection approaches for molecular detection of TORCH organisms. This review will aid in the development of a sensitive molecular assay for quick and precise detection of TORCH organisms.
Remarkably robust antiviral immune response despite combined deficiency in caspase-8 and RIPK3 Yanjun Feng, Devon Livingston-Rosanoff, Linda Roback, Aarthi Sundararajan, Samuel H. Speck, Edward S. Mocarski, Lisa P. Daley-Bauer Journal of Immunology, 2018 Caspase-8 (Casp8)–mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3–dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, Casp8−/−Ripk3−/− C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack lpr-like lymphoid hyperplasia and accumulation of either B220+CD3+ or B220−CD3+CD4+ and CD8+ T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either Casp8+/−Ripk3−/− or wild type (Casp8+/+Ripk3+/+) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.
Robust mucosal-homing antibody-secreting B cell responses induced by intramuscular administration of adjuvanted bivalent human norovirus-like particle vaccine Aarthi Sundararajan, Mark Y. Sangster, Sharon Frey, Robert L. Atmar, Wilbur H. Chen, Jennifer Ferreira, Robert Bargatze, Paul M. Mendelman, John J. Treanor, David J. Topham Vaccine, 2015 BACKGROUND: Two major antigenically heterogenous norovirus genogroups (GI and GII) commonly infect humans and are the leading cause of foodborne, viral gastrointestinal infections in adults. METHODS: We assessed B cell responses in participants in a double-blind, placebo-controlled, dose-escalation phase 1 study of the safety and immunogenicity of an intramuscular bivalent norovirus virus-like particle (VLP) vaccine. The vaccine contained a GI.1 VLP (Norwalk) and a consensus GII.4 VLP, representing the two major genotypes that cause human disease, and was administered on days 0 and 28 to healthy adults aged 18-49 years. Four separate cohorts received increasing doses of 5 μg, 15 μg, 50 μg, and 150 μg of each VLP adjuvanted in monophosphoryl lipid A and alum. PBMCs were analyzed for B cell activation and mucosal homing markers (flow cytometry) and VLP-specific and total IgG and IgA Ab-secreting cells (ASCs); and serum titers of VLP-specific IgG, IgA, and Pan-Ig were determined. RESULTS: The vaccine elicited CD27+ CD38+ plasmablasts and high frequencies of ASCs specific for both VLP antigens in the peripheral blood at 7 days after the first dose. The plasmablasts exhibited a mucosal-homing phenotype and included a high proportion of IgA ASCs. Serum antibodies increased as early as 7 days after the first immunization. CONCLUSIONS: The data suggest that a single dose of the IM bivalent norovirus vaccine is effective in activating pre-existing B cell memory. The rapid B cell response and the mucosal homing phenotype of induced ASCs are consistent with anamnestic responses in subjects primed by prior oral norovirus infection. This study is registered at ClinicalTrials.gov Identifier NCT01609257.
RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition William J. Kaiser, Lisa P. Daley-Bauer, Roshan J. Thapa, Pratyusha Mandal, Scott B. Berger, Chunzi Huang, Aarthi Sundararajan, Hongyan Guo, Linda Roback, Samuel H. Speck, John Bertin, Peter J. Gough, Siddharth Balachandran, Edward S. Mocarski Proceedings of the National Academy of Sciences of the United States of America, 2014 Significance The protein kinase receptor interacting protein 1 controls signaling via death receptors, Toll-like receptors, and retinoic acid-inducible gene 1-like receptors, dictating inflammatory outcomes as broad as cytokine activation and cell death. RIP1 makes a vital contribution during development, evident from the fact that RIP1-deficient mice die soon after birth. Here, we show that a kinase-independent function of RIP1 dampens the consequences of innate immune cell death. During parturition, RIP1 prevents the lethal consequences of RIP3-dependent necroptosis as well as caspase 8 (Casp8)-dependent apoptosis. In contrast to the RIP1-deficient phenotype, mice lacking a combination of RIP1, RIP3, and Casp8 are born and mature into viable, fertile, and immunocompetent adults. These results demonstrate the important protective role of RIP1 against physiologic and microbial death cues encountered at birth.
