Search

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern comprises several sublineages, with BA.2 and BA.2.12.1 having replaced the previously dominant BA.1 and with BA.4 and BA.5 increasing in prevalence worldwide. We show that the large number of Omicron sublineage spike mutations leads to enhanced angiotensin-converting enzyme 2 (ACE2) binding, reduced fusogenicity, and severe dampening of plasma neutralizing activity elicited by infection or seven clinical vaccines relative to the ancestral virus. Administration of a homologous or heterologous booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 across all vaccines evaluated. Our data suggest that although Omicron sublineages evade polyclonal neutralizing antibody responses elicited by primary vaccine series, vaccine boosters may provide sufficient protection against Omicron-induced severe disease.

Numerous safe and effective coronavirus disease 2019 vaccines have been developed worldwide that use various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S as compared with vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity, and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by receptor binding domain–specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies., Science Immunology, 7 (78), ISSN:2470-9468

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to acute respiratory distress syndrome (ARDS). Understanding the evolution of the virus, and immune-pathogenic processes are critical for designing future therapeutic interventions. In this review, we collate information on the structure, genome, viral life cycle, and adult and pediatric host immune responses in response to SARS-CoV-2. The immunological responses are a prototype of the developmental origins of health and disease (DOHaD) hypothesis to explain the socio-geographic differences impacting the severity and mortality rates in SARS-CoV-2 infections. The DOHaD hypothesis identifies the relevance of trained innate immunity, age groups, and geography for effective vaccinations. As COVID-19 vaccines are being rolled out, it may be pertinent to assess population-based immunological responses to understand the effectiveness and safety across different populations and age groups.

Background Pakistan introduced Ten-valent pneumococcal-conjugate-vaccine PCV10 in 2012 as a 3 + 0 schedule without catch-up. Methods Children

Background Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. Methods We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold, Molecular methods identified Shigella more commonly than microbiologic culture in younger and stunted children. A simplified clinical score containing dehydration, hospitalization, and diarrhea duration could be used to stratify vaccine trial endpoints by severity based on its ability to predict death.

Background Prolonged enteropathogen shedding after diarrhea complicates the identification of etiology in subsequent episodes and is an important driver of pathogen transmission. A standardized approach has not been applied to estimate the duration of shedding for a wide range of pathogens. Methods We used a multisite birth cohort of children 0–24 months of age from whom diarrheal and monthly nondiarrheal stools were previously tested by quantitative polymerase chain reaction for 29 enteropathogens. We modeled the probability of detection of the etiologic pathogen before and after diarrhea using a log-normal accelerated failure time survival model and estimated the median duration of pathogen carriage as well as differences in subclinical pathogen carriage 60 days after diarrhea onset in comparison to a prediarrhea baseline. Results We analyzed 3247 etiologic episodes of diarrhea for the 9 pathogens with the highest attributable burdens of diarrhea. The median duration of postdiarrheal carriage varied widely by pathogen, from about 1 week for rotavirus (median, 8.1 days [95% confidence interval {CI}, 6.2–9.6]) to >1 month for Cryptosporidium (39.5 days [95% CI, 30.6–49.0]). The largest increases in subclinical pathogen carriage before and after diarrhea were seen for Cryptosporidium (prevalence difference between 30 days prior and 60 days after diarrhea onset, 0.30 [95% CI, .23–.39]) and Shigella (prevalence difference, 0.21 [95% CI, .16–.27]). Conclusions Postdiarrheal shedding was widely variable between pathogens, with strikingly prolonged shedding seen for Cryptosporidium and Shigella. Targeted antimicrobial therapy and vaccination for these pathogens may have a relatively large impact on transmission., In a multisite birth cohort study of children 0–24 months of age and with quantitative polymerase chain reaction detection of diarrheal pathogens, we estimate the duration of postdiarrheal carriage in low-resource settings, with strikingly prolonged shedding seen for Cryptosporidium and Shigella.

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).

Background The degree of protection conferred by natural immunity is unknown for many enteropathogens, but it is important to support the development of enteric vaccines. Methods We used the Andersen-Gill extension of the Cox model to estimate the effects of previous infections on the incidence of subsequent subclinical infections and diarrhea in children under 2 using quantitative molecular diagnostics in the MAL-ED cohort. We used cross-pathogen negative control associations to correct bias due to confounding by unmeasured heterogeneity of exposure and susceptibility. Results Prior rotavirus infection was associated with a 50% lower hazard (calibrated hazard ratio [cHR], 0.50; 95% confidence interval [CI], 0.41–0.62) of subsequent rotavirus diarrhea. Strong protection was evident against Cryptosporidium diarrhea (cHR, 0.32; 95% CI, 0.20–0.51). There was also protection due to prior infections for norovirus GII (cHR against diarrhea, 0.67; 95% CI, 0.49–0.91), astrovirus (cHR, 0.62; 95% CI, 0.48–0.81), and Shigella (cHR, 0.79; 95% CI, 0.65–0.95). Minimal protection was observed for other bacteria, adenovirus 40/41, and sapovirus. Conclusions Natural immunity was generally stronger for the enteric viruses than bacteria, potentially due to less antigenic diversity. Vaccines against major causes of diarrhea may be feasible but likely need to be more immunogenic than natural infection., We estimated natural immunity to enteric pathogens as the effects of previous infections on the incidence of subsequent infections in children under 2. Prior rotavirus, Cryptosporidium, norovirus GII, astrovirus, and Shigella infections were associated with lower risk of subsequent diarrhea.

The SARS-CoV-2 Omicron variant of concern comprises three sublineages designated BA.1, BA.2, and BA.3, with BA.2 steadily replacing the globally dominant BA.1. We show that the large number of BA.1 and BA.2 spike mutations severely dampen plasma neutralizing activity elicited by infection or seven clinical vaccines, with cross-neutralization of BA.2 being consistently more potent than that of BA.1, independent of the vaccine platform and number of doses. Although mRNA vaccines induced the greatest magnitude of Omicron BA.1 and BA.2 plasma neutralizing activity, administration of a booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1 and BA.2 across all vaccines evaluated. Our data suggest that although BA.1 and BA.2 evade polyclonal neutralizing antibody responses, current vaccine boosting regimens may provide sufficient protection against Omicron-induced disease.

Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV-2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.

SUMMARYThe recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

The underperformance of oral vaccines in children of low- and middle-income countries is partly attributable to underlying environmental enteric dysfunction (EED).We conducted a longitudinal, community-based study to evaluate the association of oral rotavirus vaccine (Rotarix®) seroconversion with growth anthropometrics, EED biomarkers and intestinal enteropathogens in Pakistani infants. Children were enrolled between three to six months of their age based on their nutritional status. We measured serum anti-rotavirus immunoglobulin A (IgA) at enrollment and nine months of age with EED biomarkers and intestinal enteropathogens.A total of 391 infants received two doses of rotavirus (RV) vaccine. 331/391 provided paired blood samples. Of these 331 children, 45% seroconverted at 9 months of age, 35% did not seroconvert and 20% were seropositive at baseline. Non-seroconverted children were more likely to be stunted, wasted and underweight at enrollment. In univariate analysis, insulin-like growth factor (IGF) concentration at 6 months were higher in seroconverters, median (25th, 75th percentile): 26.3 (16.5, 43.5) ng/ml vs. 22.5 (13.6, 36.3) ng/ml for non-seroconverters, p-value = 0.024. At nine months, fecal myeloperoxidase (MPO) concentrations were significantly lower in seroconverters, 3050(1250, 7587) ng/ml vs. 4623.3 (2189, 11650) ng/ml in non-seroconverted children, p-value = 0.017. In multivariable logistic regression analysis, alpha-1 acid glycoprotein (AGP) and IGF-1 concentrations were positively associated with seroconversion at six months. The presence of sapovirus and rotavirus in fecal samples at the time of rotavirus administration, was associated with non-seroconversion and seroconversion, respectively.We detected high baseline RV seropositivity and impaired RV vaccine immunogenicity in this high-risk group of children. Healthy growth, serum IGF-1 and AGP, and fecal shedding of rotavirus were positively associated with RV IgA seroconversion following immunization, whereas the presence of sapovirus was more common in non-seroconverters.Clinical Trials ID: NCT03588013.

To address the disproportionate burden of preterm birth (PTB) in low- and middle-income countries, this study aimed to (1) verify the performance of the United States-validated spontaneous PTB (sPTB) predictor, comprised of the IBP4/SHBG protein ratio, in subjects from Bangladesh, Pakistan and Tanzania enrolled in the Alliance for Maternal and Newborn Health Improvement (AMANHI) biorepository study, and (2) discover biomarkers that improve performance of IBP4/SHBG in the AMANHI cohort. The performance of the IBP4/SHBG biomarker was first evaluated in a nested case control validation study, then utilized in a follow-on discovery study performed on the same samples. Levels of serum proteins were measured by targeted mass spectrometry. Differences between the AMANHI and U.S. cohorts were adjusted using body mass index (BMI) and gestational age (GA) at blood draw as covariates. Prediction of sPTB < 37 weeks and < 34 weeks was assessed by area under the receiver operator curve (AUC). In the discovery phase, an artificial intelligence method selected additional protein biomarkers complementary to IBP4/SHBG in the AMANHI cohort. The IBP4/SHBG biomarker significantly predicted sPTB < 37 weeks (n = 88 vs. 171 terms ≥ 37 weeks) after adjusting for BMI and GA at blood draw (AUC= 0.64, 95% CI: 0.57–0.71, p < .001). Performance was similar for sPTB < 34 weeks (n = 17 vs. 184 ≥ 34 weeks): AUC = 0.66, 95% CI: 0.51–0.82, p = .012. The discovery phase of the study showed that the addition of endoglin, prolactin, and tetranectin to the above model resulted in the prediction of sPTB < 37 with an AUC= 0.72 (95% CI: 0.66–0.79, p-value < .001) and prediction of sPTB < 34 with an AUC of 0.78 (95% CI: 0.67–0.90, p < .001). A protein biomarker pair developed in the U.S. may have broader application in diverse non-U.S. populations.

The relationship between environmental factors and child health is not well understood in rural Pakistan. This study characterized the environmental factors related to the morbidity of acute respiratory infections (ARIs), diarrhea, and growth using geographical information systems (GIS) technology. Anthropometric, address and disease prevalence data were collected through the SEEM (Study of Environmental Enteropathy and Malnutrition) study in Matiari, Pakistan. Publicly available map data were used to compile coordinates of healthcare facilities. A Pearson correlation coefficient (r) was used to calculate the correlation between distance from healthcare facilities and participant growth and morbidity. Other continuous variables influencing these outcomes were analyzed using a random forest regression model. In this study of 416 children, we found that participants living closer to secondary hospitals had a lower prevalence of ARI (r = 0.154, p &lt, 0.010) and diarrhea (r = 0.228, p &lt, 0.001) as well as participants living closer to Maternal Health Centers (MHCs): ARI (r = 0.185, p &lt, 0.002) and diarrhea (r = 0.223, p &lt, 0.001) compared to those living near primary facilities. Our random forest model showed that distance has high variable importance in the context of disease prevalence. Our results indicated that participants closer to more basic healthcare facilities reported a higher prevalence of both diarrhea and ARI than those near more urban facilities, highlighting potential public policy gaps in ameliorating rural health.

Infectious disease is the leading cause of mortality in children under five. This study has investigated environmental factors related to the morbidity of acute respiratory infections (ARIs), diarrhea, and growth using geographical information systems (GIS) technology. Anthropometric, address and disease prevalence data were collected through the SEEM study in Matiari, Pakistan. Publicly available map data was used to compile coordinates of healthcare facilities. A Pearson correlation coefficient (r) was used to calculate the correlation between distance from healthcare facilities and participant growth and morbidity. Other continuous variables influencing these outcomes were analyzed using a random forest regression model. In this study of 416 children, we found participants living closer to secondary hospitals had lower prevalence of ARI (r=0.154, p

Background Impaired 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. Method Our 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. Results We 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. Conclusion We 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.

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., Author summary EED is a subclinical condition of compromised gut integrity secondary to frequent and repeated exposure to enteropathogens in global settings with a high prevalence of undernutrition. In this study, we reported association of gut mucosal architecture with a dual sugar intestinal permeability assay (lactulose-rhamnose) in Pakistani children. In the presence of duodenal enteropathogens, features such as chronic inflammation, intra-epithelial lymphocytosis, enterocyte injury and Paneth cell reduction were consistently observed. When comparing undernourished cases and controls living in the same setting, we found urinary excretion of the sugars was similar among groups; however, variability in HAZ among children at 24 months was partially explained by a model that includes excretion values.

Background Intestinal inflammation and malabsorption in environmental enteric dysfunction (EED) are associated with early childhood growth faltering in impoverished settings worldwide. Objectives The goal of this study was to identify candidate biomarkers associated with inflammation, EED histology, and as predictors of later growth outcomes by focusing on the liver-gut axis by investigating the bile acid metabolome. Methods Undernourished rural Pakistani infants (n = 365) with weight-for-height Z score (WHZ) < –2 were followed up to the age of 24 mo and monitored for growth, infections, and EED. Well-nourished local children (n = 51) were controls, based on consistent WHZ > 0 and height-for-age Z score (HAZ) > –1 on 2 consecutive visits at 3 and 6 mo. Serum bile acid (sBA) profiles were measured by tandem MS at the ages of 3–6 and 9 mo and before nutritional intervention. Biopsies and duodenal aspirates were obtained following upper gastrointestinal endoscopy from a subset of children (n = 63) that responded poorly to nutritional intervention. BA composition in paired plasma and duodenal aspirates was compared based on the severity of EED histopathological scores and correlated to clinical and growth outcomes. Results Remarkably, >70% of undernourished Pakistani infants displayed elevated sBA concentrations consistent with subclinical cholestasis. Serum glycocholic acid (GCA) correlated with linear growth faltering (HAZ, r = –0.252 and –0.295 at the age of 3–6 and 9 mo, respectively, P

Pneumonia is the leading cause of morbidity and mortality in children worldwide. The ten valent pneumococcal vaccine (PCV10) was introduced in Pakistan's Expanded Program on Immunization (EPI) in 2012 as a 3 + 0 schedule without catchup immunization. Nasopharyngeal carriage is taken as a surrogate marker to measure the impact of pneumococcal vaccine on populations. Carriage surveys are necessary to monitor the persistence of Vaccine Type (VT) serotypes, the emergence of Non-Vaccine Type (NVT) serotypes, and their role in both transmission and disease. This article describes various troubleshooting measures which we undertook to adopt the protocol to our setting. We also used an innovative approach to describe various epidemiological parameters of vaccine effectiveness against carriage. It is important to publish these methods to allow for valid regional and temporal comparisons of results in different settings. Thus, in this article, we describe the following methods for isolating upper airway pneumococcal carriage:•Methods for the collection, transport, and storage of nasopharyngeal samples.•Methods for identification and serotyping of pneumococci.•Methods for estimation of the direct and indirect effects of pneumococcal vaccines on nasopharyngeal carriage., Graphical abstract Image, graphical abstract

Background Stunting affects up to one-third of the children in low-to-middle income countries (LMICs) and has been correlated with decline in cognitive capacity and vaccine immunogenicity. Early identification of infants at risk is critical for early intervention and prevention of morbidity. The aim of this study was to investigate patterns of growth in infants up through 48 months of age to assess whether the growth of infants with stunting eventually improved as well as the potential predictors of growth. Methods Height-for-age z-scores (HAZ) of children from Matiari (rural site, Pakistan) at birth, 18 months, and 48 months were obtained. Results of serum-based biomarkers collected at 6 and 9 months were recorded. A descriptive analysis of the population was followed by assessment of growth predictors via traditional machine learning random forest models. Results Of the 107 children who were followed up till 48 months of age, 51% were stunted (HAZ Conclusion We demonstrated that children most children with stunting at birth remained stunted at 48 months of age. Value was added for predicting growth outcomes with the use of traditional machine learning random forest models. HAZ at birth was found to be a strong predictor of subsequent growth in infants up through 48 months of age. Biomarkers of systemic inflammation, AGP, CRP, IL1, were also strong predictors of growth outcomes. These findings provide support for continued focus on interventions prenatally, at birth, and early infancy in children at risk for stunting who live in resource-constrained regions of the world.

To explore the link between altered thyroid profile and oxidative stress marker in females with unexplained infertility.The cross-sectional case-control study was carried out at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan, from June 2016 to August 2017, and comprised women aged 18-40 years regardless of ethnic background who were divided into two groups; those with unexplained infertility were the cases, while fertile women acted as the controls. Serum was analysed for triiodothyronine, thyroxine and thyroid stimulating hormone as well as for oxidative stress markers including manganese superoxide dismutase, glutathione reductase and adrenaline using enzymelinked immunosorbent assay. Data was analysed using SPSS 19.Of the 88 subjects, there were 44(50%) in each of the two groups. There was no significant difference in terms of thyroids markers except thyroxine and thyroid stimulating hormone (p0.05). There were significant differences in terms of oxidative stress markers between the groups (p0.05). A significant positive correlation of thyroid stimulating hormone was observed with manganese superoxide dismutase and adrenaline (p0.05) with a weak non significant association of glutathione reductase (p0.05).Increased thyroxine levels in females with unexplained infertility was associated with decrease in the serum levels of antioxidants.

ObjectivesStriking histopathological overlap between distinct but related conditions poses a significant disease diagnostic challenge. There is a major clinical need to develop computational methods enabling clinicians to translate heterogeneous biomedical images into accurate and quantitative diagnostics. This need is particularly salient with small bowel enteropathies; Environmental Enteropathy (EE) and Celiac Disease (CD). We built upon our preliminary analysis by developing an artificial intelligence (AI)-based image analysis platform utilizing deep learning convolutional neural networks (CNNs) for these enteropathies.MethodsData for secondary analysis was obtained from three primary studies at different sites. The image analysis platform for EE and CD was developed using convolutional neural networks (CNNs: ResNet and custom Shallow CNN). Gradient-weighted Class Activation Mappings (Grad-CAMs) were used to visualize the models’ decision making process. A team of medical experts simultaneously reviewed the stain color normalized images done for bias reduction and Grad-CAM visualizations to confirm structural preservation and biological relevance, respectively.Results461 high-resolution biopsy images from 150 children were acquired. Median age (interquartile range) was 37·5 (19·0 to 121·5) months with a roughly equal sex distribution; 77 males (51·3%). ResNet50 and Shallow CNN demonstrated 98% and 96% case-detection accuracy, respectively, which increased to 98·3% with an ensemble. Grad-CAMs demonstrated models’ ability to learn distinct microscopic morphological features.ConclusionOur AI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies which was capable of identifying biologically relevant microscopic features, emulating human pathologist decision making process, performing in the case of suboptimal computational environment, and being modified for improving disease classification accuracy. Grad-CAMs that were employed illuminated the otherwise ‘black box’ of deep learning in medicine, allowing for increased physician confidence in adopting these new technologies in clinical practice.What is knownStriking histopathological overlap exists between distinct but related conditions which poses a significant disease diagnostic challenge; such as for small bowel enteropathies including Environmental Enteropathy (EE) and Celiac Disease (CD).There is a major clinical need to develop computational [including Artificial Intelligence (AI) and deep learning] methods enabling clinicians to translate heterogeneous biomedical images into accurate and quantitative diagnostics.A major issue plaguing the use of AI in medicine is the so-called ‘black box’ of deep learning, an analogy which describes the lack of insight that humans have into how the models arrive at their decision-makingWhat is newAI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies (EE vs. CD vs. histologically normal controls).Gradient-weighted Class Activation Mappings (Grad-CAMs) illuminated the otherwise ‘black box’ of deep learning in medicine, allowing for increased physician confidence in adopting these new technologies in clinical practice.

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 (, Author summary Shigella is the second leading cause of diarrhea morbidity and mortality among children in low and middle-income countries. We characterized the epidemiology of Shigella using highly sensitive diagnostic methods in 41,405 diarrheal and monthly non-diarrheal stools from the first two years of life in a multisite birth cohort. The prevalence of Shigella varied from 4.9%-17.8% across sites, and the incidence of Shigella-attributable diarrhea was 31.8 cases (95% CI: 29.6, 34.2) per 100 child-years. Shigella diarrhea episodes were more likely to be severe and less likely to be culture positive in younger children. Older age, unimproved sanitation, low maternal education, initiating complementary foods before 3 months, and malnutrition were risk factors for Shigella. There was a linear dose-response between Shigella quantity and myeloperoxidase, a marker of intestinal inflammation, which suggests a potential mechanism for the impact of Shigella on child growth. Because culture missed most clinically relevant cases of severe diarrhea and dysentery, molecular diagnostics may be important tools in upcoming Shigella vaccine trials.

Tuberculosis (TB) in children represents a missed opportunity for diagnosis and preventive therapy. The magnitude or burden of disease in children is not fully understood due to our limitations with respect to exploring sensitive diagnostic algorithms. In a setting of TB endemicity in Pakistan, we carried out a proof-of-concept study to evaluate for the first time the performance of B cell analyses by the use of well-defined diagnostic criteria and NIH consensus guidelines as “culture-confirmed,” “probable,” and “possible” TB groups. In contrast to detection of serum antibody, we focused on mycobacterial-antibody-secreting cell (MASC) detection as a marker of active disease in children with a strong suspicion of TB. Further work exploring a larger panel of inflammatory biomarkers and enrichment of B cells with the objective of increasing the sensitivity of the current MASC assay would lead to the development of a field-friendly assay for timely diagnosis of childhood TB., 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

Despite nutrition interventions, stunting thought to be secondary to underlying environmental enteropathy (EE) remains pervasive among infants residing in resource-poor countries and remains poorly characterized. From a birth cohort of 380 children, 65 malnourished infants received 12 weeks of nutritional supplementation with ready-to-use therapeutic food (RUTF). Eleven children with insufficient response to RUTF underwent upper endoscopy with duodenal biopsies, which were compared with U.S., age-matched specimens for healthy, celiac disease, non-celiac villous atrophy, non-celiac intraepithelial lymphocytosis, and graft-versus-host disease patients. Of the 11 children biopsied, EE was found in 10 (91%) with one subject with celiac disease. Morphometry demonstrated decreased villus-to-crypt (V:C) ratios in EE relative to healthy and non-celiac lymphocytosis patients. Environmental enteropathy villus volumes were significantly decreased relative to healthy controls. In EE, average CD3+ cells per 100 epithelial cells and per 1,000 µm2 of lamina propria and the number of lamina propria CD20+ B-cell aggregates were increased relative to all other groups. Our results indicate that V:C ratios are reduced in EE but are less severe than in celiac disease. Environmental enteropathy intraepithelial and lamina propria T lymphocytosis is of greater magnitude than that in celiac disease. The increases in lamina propria B and T lymphocytes suggest that non-cytolytic lymphocytic activation may be a more prominent feature of EE relative to celiac disease. These results provide new insights into shared yet distinct histological and immunological features of EE and celiac disease in children.

Environmental Enteric Dysfunction (EED), a syndrome characterized by chronic gut inflammation, contributes towards stunting and poor response to enteric vaccines in children in developing countries. In this study, we evaluated major putative biomarkers of EED using growth faltering as its clinical proxy. Newborns (n = 380) were enrolled and followed till 18 months with monthly anthropometry. Biomarkers associated with gut and systemic inflammation were assessed at 6 and 9 months. Linear mixed effects model was used to determine the associations of these biomarkers with growth faltering between birth and 18 months. Fecal myeloperoxidase (neutrophil activation marker) at 6 months [β = −0.207, p = 0.005], and serum GLP 2 (enterocyte proliferation marker) at 6 and 9 months [6M: β = −0.271, p = 0.035; 9M: β = −0.267, p = 0.045] were associated with decreasing LAZ score. Ferritin at 6 and 9 months was associated with decreasing LAZ score [6M: β = −0.882, p

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.

The Study of Environmental Enteropathy (EE) and Malnutrition in Pakistan (SEEM) is a community intervention trial designed to understand the pathophysiology of EE and its associated clinical phenotypes in children less than two years of age. Undernutrition is the underlying cause of three million deaths annually, accounting for 45% of all deaths among children under five years of age. Linear growth stunting affects a further 165 million. In Pakistan, where our study is focused, an estimated one in three children are underweight and nearly half are stunted. This manuscript summarizes the challenges faced by the field team and the solutions employed for achieving our research objectives at the rural field site, Matiari. Our study design involved a longitudinal collection of field data and biological samples which required constant engagement of field staff with the participating families. Challenges faced in SEEM included: 1) quality assurance parameters of anthropometry data; 2) 24-hour food recall data collection; 3) clinical management of acutely sick children enrolled in our study; 4) obtaining informed consent for upper gastrointestinal endoscopy (an invasive procedure); and 5) biological sample collection (blood, urine, stool) from malnourished children at the community level. We accomplished our goal of enrolling 350 malnourished children and 50 well-nourished children with a more than 95% success rate of collecting blood, urine and stool specimens. We collected 72% microbiome stool samples within 30 minutes of the child passing stool. We conducted an upper gastrointestinal biopsy in 63 children. Broadly, solutions to these challenges included good governance, the establishment of credibility, and the development of a longitudinal bidirectional relationship embedded in trust and effective communication with the caretakers of the children enrolled in our study. Continuous monitoring, regular training of staff, and effective teamwork were key factors that led to the successful execution of our study.

Background A major limitation to understanding the etiopathogenesis of environmental enteric dysfunction (EED) is the lack of a comprehensive, reproducible histologic framework for characterizing the small bowel lesions. We hypothesized that the development of such a system will identify unique histology features for EED, and that some features might correlate with clinical severity. Methods Duodenal endoscopic biopsies from two cohorts where EED is prevalent (Pakistan, Zambia) and North American children with and without gluten sensitive enteropathy (GSE) were processed for routine hematoxylin & eosin (H&E) staining, and scanned to produce whole slide images (WSIs) which we shared among study pathologists via a secure web browser-based platform. A semi-quantitative scoring index composed of 11 parameters encompassing tissue injury and response patterns commonly observed in routine clinical practice was constructed by three gastrointestinal pathologists, with input from EED experts. The pathologists then read the WSIs using the EED histology index, and inter-observer reliability was assessed. The histology index was further used to identify within- and between-child variations as well as features common across and unique to each cohort, and those that correlated with host phenotype. Results Eight of the 11 histologic scoring parameters showed useful degrees of variation. The overall concordance across all parameters was 96% weighted agreement, kappa 0.70, and Gwet’s AC 0.93. Zambian and Pakistani tissues shared some histologic features with GSE, but most features were distinct, particularly abundance of intraepithelial lymphocytes in the Pakistani cohort, and marked villous destruction and loss of secretory cell lineages in the Zambian cohort. Conclusions We propose the first EED histology index for interpreting duodenal biopsies. This index should be useful in future clinical and translational studies of this widespread, poorly understood, and highly consequential disorder, which might be caused by multiple contributing processes, in different regions of the world., Author summary The study of EED has been limited by the lack of a rigorously tested, reproducible histology index that can provide insight to the pathogenesis of this entity. In this study we report the first duodenal histology index that was developed using an unbiased approach, with excellent inter-observer reproducibility, for the study of EED. The EED histology index readily identified histologic features that are common or unique to cohorts of distinct geographic locations. Incorporating the histology index into future clinical studies will provide useful insight into the pathogenesis and for intervention strategy development.

Background Few studies have evaluated the association between preexisting vitamin D deficiency and incident tuberculosis (TB). We assessed the impact of baseline vitamins D levels on TB disease risk. Methods and findings We assessed the association between baseline vitamin D and incident TB in a prospective cohort of 6,751 HIV-negative household contacts of TB patients enrolled between September 1, 2009, and August 29, 2012, in Lima, Peru. We screened for TB disease at 2, 6, and 12 months after enrollment. We defined cases as household contacts who developed TB disease at least 15 days after enrollment of the index patient. For each case, we randomly selected four controls from among contacts who did not develop TB disease, matching on gender and year of age. We also conducted a one-stage individual-participant data (IPD) meta-analysis searching PubMed and Embase to identify prospective studies of vitamin D and TB disease until June 8, 2019. We included studies that assessed vitamin D before TB diagnosis. In the primary analysis, we defined vitamin D deficiency as 25–(OH)D < 50 nmol/L, insufficiency as 50–75 nmol/L, and sufficiency as >75nmol/L. We estimated the association between baseline vitamin D status and incident TB using conditional logistic regression in the Lima cohort and generalized linear mixed models in the meta-analysis. We further defined severe vitamin D deficiency as 25–(OH)D < 25 nmol/L and performed stratified analyses by HIV status in the IPD meta-analysis. In the Lima cohort, we analyzed 180 cases and 709 matched controls. The adjusted odds ratio (aOR) for TB risk among participants with baseline vitamin D deficiency compared to sufficient vitamin D was 1.63 (95% CI 0.75–3.52; p = 0.22). We included seven published studies in the meta-analysis and analyzed 3,544 participants. In the pooled analysis, the aOR was 1.48 (95% CI 1.04–2.10; p = 0.03). The aOR for severe vitamin D deficiency was 2.05 (95% CI 0.87–4.87; p trend for decreasing 25–(OH)D levels from sufficient vitamin D to severe deficiency = 0.02). Among 1,576 HIV-positive patients, vitamin D deficiency conferred a 2-fold (aOR 2.18, 95% CI 1.22–3.90; p = 0.01) increased risk of TB, and the aOR for severe vitamin D deficiency compared to sufficient vitamin D was 4.28 (95% CI 0.85–21.45; p = 0.08). Our Lima cohort study is limited by the short duration of follow-up, and the IPD meta-analysis is limited by the number of possible confounding covariates available across all studies. Conclusion Our findings suggest vitamin D predicts TB disease risk in a dose-dependent manner and that the risk of TB disease is highest among HIV-positive individuals with severe vitamin D deficiency. Randomized control trials are needed to evaluate the possible role of vitamin D supplementation on reducing TB disease risk., Megan B. Murray and colleagues combine data from a case-control study in Lima, Peru, with previously published prospective studies to investigate whether low vitamin D is associated with risk of developing tuberculosis disease., Author summary Why was this study done? Although multiple lines of evidence suggest that vitamin D may play a role in host susceptibility to tuberculosis (TB) disease, the impact of low vitamin D on the risk of developing TB disease has not yet been firmly established. What did the researchers do and find? We conducted a study in Lima, Peru, in which we measured serum 25–(OH)D levels in individuals at high risk for TB disease and followed them for development of TB over 1 year. We pooled individual-level data from seven previously published prospective studies conducted worldwide and from our Lima cohort. We found that individuals with low levels of vitamin D were at higher risk of future progression to TB disease. What do these findings mean? These findings suggest that vitamin D deficiency is a risk factor for developing TB disease. Randomized control trials are needed to determine whether vitamin D supplementation reduces risk of TB disease.

Environmental Enteric Dysfunction (EED) is an acquired small intestinal inflammatory condition underlying high rates of stunting in children