Search

Abstract Background Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP). Methods HG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell – IFN-γ activation was assessed by ELISpot. Results Overall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike. Conclusions Antibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population.

Abstract The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinase CTX-M and carbapenemases KPC, IMP, NDM, and VIM, remain taxonomically restricted to Proteobacteria. Even cfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted to Bacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted to Bacteroides species, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota.

BackgroundPakistan is endemic to a diverse set of parasitic, mycobacterial and viral diseases. The recognition of BCG Trained Immunity (TI) led us to postulate that the continued presence of BCG-TI may play a protective role, previously reported for both infectious and noninfectious conditions. Most of the previous studies have addressed the issue of BCG-TI in the paediatric populations. This study addressed the key issue of maintenance of BCG-TI in a wider age range (adolescent and adults) to identify the strength and quality of the immune responses.ObjectiveTo assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections.Study designIn this cross-sectional study, healthy young adults and adolescents (n = 20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition.MethodsBCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network [macrophages [M1 (TNFα, IFNγ), M2 (IL10)], NK (IL2), Gamma delta (γδ) T (IL17, IL4)] and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay.ResultsSignificant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNFα (9148 pg/ml) followed by IL10 (488 pg/ml) and IFNγ (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNFα, 40 fold fo r IL10, and 27 fold for IFNγ. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG.ConclusionThe presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro- and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.

Abstract Background Early childhood caries poses a significant health issue in children under 6 years old. It is determined that Streptococcus mutans is a primary etiological agent, likely to be transferred through maternal contact. Objectives To determine the association of maternal S. mutans counts with S. mutans counts in their children between 6 and 30 months of age, and to determine the maternal and child DMFT (decayed, missing, and filled teeth) indices. Material and Methods A community‐based cross‐sectional study was conducted in Karachi, Pakistan. A sample of 193 dyads of mother–children (6–30 months of age) was selected via purposive sampling. Saliva samples of the dyads were collected to assess S. mutans count. Caries assessment was performed for both using the DMFT index. A pretested questionnaire was used. The association of bottle‐feeding, oral hygiene measures, and other factors with S. mutans counts in children were also explored. Zero‐inflated negative binomial regression model at a 5% level of significance was applied using STATA version 12.0. Results Out of 193 children, 109 (56.47%) were males and 84 (43.52%) were females. The mean age of mothers and children was 29.4 ± 6.2 years and 19.54 ± 6.8 months, respectively. Maternal S. mutans counts were not statistically associated with child's S. mutans counts (Mean child's S. mutans count ratio: 1; 95% confidence interval [CI]: 1, 1.01; p = .882). Compared with children who were breastfed, S. mutans counts were higher in children who were bottle‐fed (mean S. mutans count ratio= 4.85 [95% CI: 1.53, 15.41], p = .007). Age of mother and present caries status of mothers was significantly associated with the child's S. mutans count. Conclusion No association between maternal S. mutans and child S. mutans was observed. However, maternal age, children who were breastfed, children who did not use pacifiers, and children with mothers who did not have caries, exhibited low S. mutans counts in their saliva.

IntroductionEnvironmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy.MethodsA community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3–6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens.ResultsGiardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO).ConclusionProtozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering.

Abstract Objectives 1- To compare the effectiveness of 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline and a novel solution Neem extract (Azardirachta indica) in reducing intra-oral viral load in COVID-19 positive patients. 2- To determine the salivary cytokine profiles of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL- 17 among COVID-19 patients subjected to 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline or Neem extract (Azardirachta indica) based gargles. Trial design This will be a parallel group, quadruple blind-randomised controlled pilot trial with an add on laboratory based study. Participants A non-probability, purposive sampling technique will be followed to identify participants for this study. The clinical trial will be carried out at the Aga Khan University Hospital (AKUH), Karachi, Pakistan. The viral PCR tests will be done at main AKUH clinical laboratories whereas the immunological tests (cytokine analysis) will be done at the Juma research laboratory of AKUH. The inclusion criteria are laboratory-confirmed COVID-19 positive patients, male or female, in the age range of 18-65 years, with mild to moderate disease, already admitted to the AKUH. Subjects with low Glasgow coma score, with a history of radiotherapy or chemotherapy, who are more than 7 days past the onset of COVID- 19 symptoms, or intubated or edentulous patients will be excluded. Patients who are being treated with any form of oral or parenteral antiviral therapy will be excluded, as well as patients with known pre-existing chronic mucosal lesions such as lichen planus. Intervention and comparator Group A (n=10) patients on 10 ml gargle and nasal lavage using 0.2% Povidone-Iodine (Betadiene® by Aviro Health Inc./ Pyodine® by Brooks Pharma Inc.) for 20-30 seconds, thrice daily for 6 days. Group B (n=10) patients will be subjected to 10 ml gargle and nasal lavage using 1% Hydrogen peroxide (HP® by Karachi Chemicals Products Inc./ ActiveOxy® by Boumatic Inc.) for 20-30 seconds, thrice daily for 6 days. Group C will comprised of (n=10) subjects on 10ml gargle and nasal lavage using Neem extract solution (Azardirachta indica) formulated by Karachi University (chemistry department laboratories) for 20-30 seconds, thrice daily for 6 days. Group D (n=10) patients will use 2% hypertonic saline (Plabottle® by Otsuka Inc.) gargle and nasal lavage for a similar time period. Group E (n=10) will serve as positive controls. These will be given simple distilled water gargles and nasal lavage for 20-30 seconds, thrice daily for six days. For nasal lavage, a special douche syringe will be provided to each participant. Its use will be thoroughly explained by the data collection officer. After each use, the patient is asked not to eat, drink, or rinse their mouth for the next 30 minutes. Main outcomes The primary outcome is the reduction in the intra-oral viral load confirmed with real time quantitative PCR. Randomisation The assignment to the study group/ allocation will be done using the sealed envelope method under the supervision of Clinical Trial Unit (CTU) of Aga Khan University, Karachi, Pakistan. The patients will be randomised to their respective study group (1:1:1:1:1 allocation ratio) immediately after the eligibility assessment and consent administration is done. Blinding (masking) The study will be quadruple-blinded. Patients, intervention provider, outcome assessor and the data collection officer will be blinded. The groups will be labelled as A, B, C, D or E. The codes of the intervention will be kept in lock & key at the CTU and will only be revealed at the end of study or if the study is terminated prematurely. Numbers to be randomised (sample size) As there is no prior work on this research question, so no assumptions for the sample size calculation could be made. The present study will serve as a pilot trial. We intend to study 50 patients in five study groups with 10 patients in each study group. For details, please refer to Fig. 1 for details. Trial Status Protocol version is 7.0, approved by the department and institutional ethics committees and clinical trial unit of the university hospital. Recruitment is planned to start as soon as the funding is sanctioned. The total duration of the study is expected to be 6 months i.e. August 2020-January 2021. Trial registration This study protocol was registered at www.clinicaltrials.gov on 10 April 2020 NCT04341688 . Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). Fig. 1 Flow diagram of study-participants’ timeline

Bifidobacterium longum subspecies detected in infant stool have been associated with numerous subsequent health outcomes and are potential early markers of deviation from healthy developmental trajectories. This analysis derived indicators of carriage and early colonization with B. infantis and B. longum and quantified their associations with a panel of early-life exposures and outcomes. In a sub-study nested within a multi-site birth cohort, extant stool samples from infants in Bangladesh, Pakistan and Tanzania were tested for presence and quantity of two Bifidobacterium longum subspecies. The results were matched to indicators of nutritional status, enteropathogen infection, histo-blood group antigens, vaccine response and feeding status and regression models were fitted to test for associations while adjusting for covariates. B. infantis was associated with lower quantity of and decreased odds of colonization with B. longum, and vice versa. Length at birth was associated with a 0.36 increase in log10B. infantis and a 0.28 decrease in B. longum quantity at 1 month of age. B. infantis colonization was associated with fewer viral infections and small reductions in the risk of rotavirus and sapovirus infections, but not reduced overall diarrheal disease risk. No associations with vaccine responses, HBGAs or later nutritional status were identified. Suboptimal intrauterine growth and a shorter duration of exclusive breastfeeding may predispose infants to early intestinal colonization with the B. longum subspecies at the expense of B. infantis, thus denying them potential benefits of reduced enteric virus episodes.

Environmental enteric dysfunction (EED) is characterized by intestinal inflammation, malabsorption and growth-faltering in children with heightened exposure to gut pathogens. The aim of this study was to characterize serum non-esterified fatty acids (NEFA), in association with childhood undernutrition and EED, as potential biomarkers to predict growth outcomes. The study comprised a cohort of undernourished rural Pakistani infants (n = 365) and age-matched controls followed prospectively up to 24 months of age. Serum NEFA were quantified at ages 3–6 and 9 months and correlated with growth outcomes, serum bile acids and EED histopathological biomarkers. Serum NEFA correlated with linear growth-faltering and systemic and gut biomarkers of EED. Undernourished children exhibited essential fatty acid deficiency (EFAD), with low levels of linoleic acid and total n-6 polyunsaturated fatty acids, compensated by increased levels of oleic acid and increased elongase and desaturase activities. EFAD correlated with reduced anthropometric Z scores at 3–6 and 9 months of age. Serum NEFA also correlated with elevated BA and liver dysfunction. Essential fatty acid depletion and altered NEFA metabolism were highly prevalent and associated with acute and chronic growth-faltering in EED. The finding suggests that targeting early interventions to correct EFAD and promote FA absorption in children with EED may facilitate childhood growth in high-risk settings.

Environmental enteric dysfunction (EED) is a subclinical condition of intestinal inflammation, barrier dysfunction and malabsorption associated with growth faltering in children living in poverty. This study explores association of altered duodenal permeability (lactulose, rhamnose and their ratio) with higher burden of enteropathogen in the duodenal aspirate, altered histopathological findings and higher morbidity (diarrhea) that is collectively associated with linear growth faltering in children living in EED endemic setting. In a longitudinal birth cohort, 51 controls (WHZ > 0, HAZ > -1.0) and 63 cases (WHZ< -2.0, refractory to nutritional intervention) were recruited. Anthropometry and morbidity were recorded on monthly bases up to 24 months of age. Dual sugar assay of urine collected after oral administration of lactulose and rhamnose was assessed in 96 children from both the groups. Duodenal histopathology (n = 63) and enteropathogen analysis of aspirate via Taqman array card (n = 60) was assessed in only cases. Giardia was the most frequent pathogen and was associated with raised L:R ratio (p = 0.068). Gastric microscopy was more sensitive than duodenal aspirate in H. pylori detection. Microscopically confirmed H. pylori negatively correlated with HAZ at 24 months (r = -0.313, p = 0.013). Regarding histopathological parameters, goblet cell reduction significantly correlated with decline in dual sugar excretion (p< 0.05). Between cases and controls, there were no significant differences in the median (25th, 75th percentile) of urinary concentrations (μg/ml) of lactulose [27.0 (11.50, 59.50) for cases vs. 38.0 (12.0, 61.0) for controls], rhamnose [66.0 (28.0, 178.0) vs. 86.5 (29.5, 190.5)] and L:R ratio [0.47 (0.24, 0.90) vs. 0.51 (0.31, 0.71)] respectively. In multivariable regression model, 31% of variability in HAZ at 24 months of age among cases and controls was explained by final model including dual sugars. In conclusion, enteropathogen burden is associated with altered histopathological features and intestinal permeability. In cases and controls living in settings of endemic enteropathy, intestinal permeability test may predict linear growth. However, for adoption as a screening tool for EED, further validation is required due to its complex intestinal pathophysiology.

Stunting is a global public health issue. We sought to train and evaluate machine learning (ML) classification algorithms on the Zambia Demographic Health Survey (ZDHS) dataset to predict stunting among children under the age of five in Zambia. We applied Logistic regression (LR), Random Forest (RF), SV classification (SVC), XG Boost (XgB) and Naïve Bayes (NB) algorithms to predict the probability of stunting among children under five years of age, on the 2018 ZDHS dataset. We calibrated predicted probabilities and plotted the calibration curves to compare model performance. We computed accuracy, recall, precision and F1 for each machine learning algorithm. About 2327 (34.2%) children were stunted. Thirteen of fifty-eight features were selected for inclusion in the model using random forest. Calibrating the predicted probabilities improved the performance of machine learning algorithms when evaluated using calibration curves. RF was the most accurate algorithm, with an accuracy score of 79% in the testing and 61.6% in the training data while Naïve Bayesian was the worst performing algorithm for predicting stunting among children under five in Zambia using the 2018 ZDHS dataset. ML models aids quick diagnosis of stunting and the timely development of interventions aimed at preventing stunting.

BackgroundImpaired growth among infants remains one of the leading nutrition problems globally. In this study, we aimed to compare the growth trajectory rate and evaluate growth trajectory characteristics among children, who are HIV exposed uninfected (HEU) and HIV unexposed uninfected (HUU), under two years in Zambia.MethodOur study used data from the ROVAS II study (PACTR201804003096919), an open-label randomized control trial of two verses three doses of live, attenuated, oral RotarixTM administered 6 &10 weeks or at 6 &10 weeks plus an additional dose at 9 months of age, conducted at George clinic in Lusaka, Zambia. Anthropometric measurements (height and weight) were collected on all scheduled and unscheduled visits. We defined linear growth velocity as the rate of change in height and estimated linear growth velocity as the first derivative of the mixed effect model with fractional polynomial transformations and, thereafter, used the second derivative test to determine the peak height and age at peak heigh.ResultsWe included 212 infants in this study with median age 6 (IQR: 6-6) weeks of age. Of these 97 (45.3%) were female, 35 (16.4%) were stunted, and 59 (27.6%) were exposed to HIV at baseline. Growth velocity was consistently below the 3rd percentile of the WHO linear growth standard for HEU and HUU children. The peak height and age at peak height among HEU children were 74.7 cm (95% CI = 73.9-75.5) and 15.5 months (95% CI = 14.7-16.3) respectively and those for HUU were 73 cm (95% CI = 72.1-74.0) and 15.6 months (95% CI = 14.5-16.6) respectively.ConclusionWe found no difference in growth trajectories between infants who are HEU and HUU. However, the data suggests that poor linear growth is universal and profound in this cohort and may have already occurred in utero.

Culture-independent diagnostics have revealed a larger burden of Shigella among children in low-resource settings than previously recognized. We further characterized the epidemiology of Shigella in the first two years of life in a multisite birth cohort. We tested 41,405 diarrheal and monthly non-diarrheal stools from 1,715 children for Shigella by quantitative PCR. To assess risk factors, clinical factors related to age and culture positivity, and associations with inflammatory biomarkers, we used log-binomial regression with generalized estimating equations. The prevalence of Shigella varied from 4.9%-17.8% in non-diarrheal stools across sites, and the incidence of Shigella-attributable diarrhea was 31.8 cases (95% CI: 29.6, 34.2) per 100 child-years. The sensitivity of culture compared to qPCR was 6.6% and increased to 27.8% in Shigella-attributable dysentery. Shigella diarrhea episodes were more likely to be severe and less likely to be culture positive in younger children. Older age (RR: 1.75, 95% CI: 1.70, 1.81 per 6-month increase in age), unimproved sanitation (RR: 1.15, 95% CI: 1.03, 1.29), low maternal education (

ABSTRACT Reliance on microbiologic methods to diagnose Mycobacterium tuberculosis infection is a suboptimal approach for children due in part to the paucibacillary nature of the disease. A blood-based biomarker assay, such as the mycobacterial-antibody-secreting cell (MASC) assay, could be a major advance for the field of study of pediatric tuberculosis (TB). Children

Background: Viral flu is the predominant cause of hospitalization in young children, which invariably leads to enhanced morbidity and mortality in children in developing countries. Initial treatment of viral flu is based on presumptive diagnosis. Bedside testing is not common in clinical settings because of variable sensitivity and specificity of rapid tests in different settings. Methods: To address this issue, we evaluated the performance of Binax influenza A/B rapid testing kit against two robust molecular platforms (quantitative real-time polymerase chain reaction [qRT-PCR] and TaqMan array card [TAC]) in 24 nasopharyngeal (NP) swabs, collected from children under 5 years of age. Results: Binax was found to be less sensitive (56%), but 100% specific compared to qRT-PCR (100%) and TAC (>100%). Using TAC cards, 75% of samples were found to be coinfected with other bacterial and viral targets. Conclusion: Binax flu is suitable for bedside testing in clinical and community settings. The negative results of Binax should be interpreted with caution and confirmed by rapid molecular tests.

Objective/Background: The characterization of tuberculosis (TB) patients as slow or fast responders post anti-TB treatment has always been a matter of tremendous interest as slow responders are most likely to relapse and/or develop complications. Pulmonary tissue healing as assessed with radiology is the only available tool for tissue recovery but is not predictive at intake. The objective of the current study was to assess biomarkers associated with fast and slow recovery in TB patients at recruitment. Methods: Pulmonary TB patients (N = 15) were assessed for radiological recovery serially in parallel with clinical signs and symptoms, hematological parameters, and plasma cytokines at 0months, 6months, 12months, and 24months. On the basis of differential radiological healing, patients were characterized into slow (>12 months), intermediate (