Influence of hydrometeorological risk factors on child diarrhea and enteropathogens in rural Bangladesh.

BACKGROUND: A number of studies have detected relationships between weather and diarrhea. Few have investigated associations with specific enteric pathogens. Understanding pathogen-specific relationships with weather is crucial to inform public health in low-resource settings that are especially vulnerable to climate change. OBJECTIVES: Our objectives were to identify weather and environmental risk factors associated with diarrhea and enteropathogen prevalence in young children in rural Bangladesh, a population with high diarrheal disease burden and vulnerability to weather shifts under climate change. METHODS: We matched temperature, precipitation, surface water, and humidity data to observational longitudinal data from a cluster-randomized trial that measured diarrhea and enteropathogen prevalence in children 6 months-5.5 years from 2012-2016. We fit generalized additive mixed models with cubic regression splines and restricted maximum likelihood estimation for smoothing parameters. RESULTS: Comparing weeks with 30°C versus 15°C average temperature, prevalence was 3.5% higher for diarrhea, 7.3% higher for Shiga toxin-producing Escherichia coli (STEC), 17.3% higher for enterotoxigenic E. coli (ETEC), and 8.0% higher for Cryptosporidium. Above-median weekly precipitation (median: 13mm; range: 0-396mm) was associated with 29% higher diarrhea (adjusted prevalence ratio 1.29, 95% CI 1.07, 1.55); higher Cryptosporidium, ETEC, STEC, Shigella, Campylobacter, Aeromonas, and adenovirus 40/41; and lower Giardia, sapovirus, and norovirus prevalence. Other associations were weak or null. DISCUSSION: Higher temperatures and precipitation were associated with higher prevalence of diarrhea and multiple enteropathogens; higher precipitation was associated with lower prevalence of some enteric viruses. Our findings emphasize the heterogeneity of the relationships between hydrometeorological variables and specific enteropathogens, which can be masked when looking at composite measures like all-cause diarrhea. Our results suggest that preventive interventions targeted to reduce enteropathogens just before and during the rainy season may more effectively reduce child diarrhea and enteric pathogen carriage in rural Bangladesh and in settings with similar meteorological characteristics, infrastructure, and enteropathogen transmission.

Treatment Heterogeneity of Water, Sanitation, Hygiene, and Nutrition Interventions on Child Growth by Environmental Enteric Dysfunction and Pathogen Status for Young Children in Bangladesh.

Background: Water, sanitation, hygiene (WSH), nutrition (N), and combined (N+WSH) interventions are often implemented by global health organizations, but WSH interventions may insufficiently reduce pathogen exposure, and nutrition interventions may be modified by environmental enteric dysfunction (EED), a condition of increased intestinal permeability and inflammation. This study investigated the heterogeneity of these treatments' effects based on individual pathogen and EED biomarker status with respect to child linear growth. Methods: We applied cross-validated targeted maximum likelihood estimation and super learner ensemble machine learning to assess the conditional treatment effects in subgroups defined by biomarker and pathogen status. We analyzed treatment (N+WSH, WSH, N, or control) randomly assigned in-utero, child pathogen and EED data at 14 months of age, and child LAZ at 28 months of age. We estimated the difference in mean child length for age Z-score (LAZ) under the treatment rule and the difference in stratified treatment effect (treatment effect difference) comparing children with high versus low pathogen/biomarker status while controlling for baseline covariates. Results: We analyzed data from 1,522 children, who had median LAZ of -1.56. We found that myeloperoxidase (N+WSH treatment effect difference 0.0007 LAZ, WSH treatment effect difference 0.1032 LAZ, N treatment effect difference 0.0037 LAZ) and Campylobacter infection (N+WSH treatment effect difference 0.0011 LAZ, WSH difference 0.0119 LAZ, N difference 0.0255 LAZ) were associated with greater effect of all interventions on growth. In other words, children with high myeloperoxidase or Campylobacter infection experienced a greater impact of the interventions on growth. We found that a treatment rule that assigned the N+WSH (LAZ difference 0.23, 95% CI (0.05, 0.41)) and WSH (LAZ difference 0.17, 95% CI (0.04, 0.30)) interventions based on EED biomarkers and pathogens increased predicted child growth compared to the randomly allocated intervention. Conclusions: These findings indicate that EED biomarker and pathogen status, particularly Campylobacter and myeloperoxidase (a measure of gut inflammation), may be related to impact of N+WSH, WSH, and N interventions on child linear growth.

Systemic inflammation, enteropathogenic E. Coli, and micronutrient insufficiencies in the first trimester as possible predictors of preterm birth in rural Bangladesh: a prospective study.

BACKGROUND: An incomplete understanding of preterm birth is especially concerning for low-middle income countries, where preterm birth has poorer prognoses. While systemic proinflammatory processes are a reportedly normal component of gestation, excessive inflammation has been demonstrated as a risk factor for preterm birth. There is minimal research on the impact of excessive maternal inflammation in the first trimester on the risk of preterm birth in low-middle income countries specifically. METHODS: Pregnant women were enrolled at the rural Bangladesh site of the National Institute of Child Health Global Network Maternal Newborn Health Registry. Serum samples were collected to measure concentrations of the inflammatory markers C-reactive protein (CRP) and Alpha-1-acid glycoprotein (AGP), and stool samples were collected and analyzed for enteropathogens. We examined associations of maternal markers in the first-trimester with preterm birth using logistic regression models. CRP and AGP were primarily modeled with a composite inflammation predictor. RESULTS: Out of 376 singleton births analyzed, 12.5% were preterm. First trimester inflammation was observed in 58.8% of all births, and was significantly associated with increased odds of preterm birth (adjusted odds ratio [aOR] = 2.23; 95% confidence interval [CI]: 1.03, 5.16), independent of anemia. Maternal vitamin B12 insufficiency (aOR = 3.33; 95% CI: 1.29, 8.21) and maternal anemia (aOR = 2.56; 95% CI: 1.26, 5.17) were also associated with higher odds of preterm birth. Atypical enteropathogenic E. coli detection showed a significant association with elevated AGP levels and was significantly associated with preterm birth (odds ratio [OR] = 2.36; 95% CI: 1.21, 4.57), but not associated with CRP. CONCLUSIONS: Inflammation, anemia, and vitamin B12 insufficiency in the first trimester were significantly associated with preterm birth in our cohort from rural Bangladesh. Inflammation and anemia were independent predictors of premature birth in this low-middle income setting where inflammation during gestation was widespread. Further research is needed to identify if infections such as enteropathogenic E. coli are a cause of inflammation in the first trimester, and if intervention for infection would decrease preterm birth.

Interactive SARS-CoV-2 dashboard for real-time geospatial visualisation of sewage and clinical surveillance data from Dhaka, Bangladesh: a tool for public health situational awareness.

Throughout the COVID-19 pandemic, many dashboards were created to visualise clinical case incidence. Other dashboards have displayed SARS-CoV-2 sewage data, largely from countries with formal sewage networks. However, very few dashboards from low-income and lower-middle-income countries integrated both clinical and sewage data sets. We created a dashboard to track in real-time both COVID-19 clinical cases and the level of SARS-CoV-2 virus in sewage in Dhaka, Bangladesh. The development of this dashboard was a collaborative iterative process with Bangladesh public health stakeholders to include specific features to address their needs. The final dashboard product provides spatiotemporal visualisations of COVID-19 cases and SARS-CoV-2 viral load at 51 sewage collection sites in 21 wards in Dhaka since 24 March 2020. Our dashboard was updated weekly for the Bangladesh COVID-19 national task force to provide supplemental data for public health stakeholders making public policy decisions on mitigation efforts. Here, we highlight the importance of working closely with public health stakeholders to create a COVID-19 dashboard for public health impact. In the future, the dashboard can be expanded to track trends of other infectious diseases as sewage surveillance is increased for other pathogens.

Real-time sewage surveillance for SARS-CoV-2 in Dhaka, Bangladesh versus clinical COVID-19 surveillance: a longitudinal environmental surveillance study (December, 2019-December, 2021).

BACKGROUND: Clinical surveillance for COVID-19 has typically been challenging in low-income and middle-income settings. From December, 2019, to December, 2021, we implemented environmental surveillance in a converging informal sewage network in Dhaka, Bangladesh, to investigate SARS-CoV-2 transmission across different income levels of the city compared with clinical surveillance. METHODS: All sewage lines were mapped, and sites were selected with estimated catchment populations of more than 1000 individuals. We analysed 2073 sewage samples, collected weekly from 37 sites, and 648 days of case data from eight wards with varying socioeconomic statuses. We assessed the correlations between the viral load in sewage samples and clinical cases. FINDINGS: SARS-CoV-2 was consistently detected across all wards (low, middle, and high income) despite large differences in reported clinical cases and periods of no cases. The majority of COVID-19 cases (26 256 [55·1%] of 47 683) were reported from Ward 19, a high-income area with high levels of clinical testing (123 times the number of tests per 100 000 individuals compared with Ward 9 [middle-income] in November, 2020, and 70 times the number of tests per 100 000 individuals compared with Ward 5 [low-income] in November, 2021), despite containing only 19·4% of the study population (142 413 of 734 755 individuals). Conversely, a similar quantity of SARS-CoV-2 was detected in sewage across different income levels (median difference in high-income vs low-income areas: 0·23 log10 viral copies + 1). The correlation between the mean sewage viral load (log10 viral copies + 1) and the log10 clinical cases increased with time (r = 0·90 in July-December, 2021 and r=0·59 in July-December, 2020). Before major waves of infection, viral load quantity in sewage samples increased 1-2 weeks before the clinical cases. INTERPRETATION: This study demonstrates the utility and importance of environmental surveillance for SARS-CoV-2 in a lower-middle-income country. We show that environmental surveillance provides an early warning of increases in transmission and reveals evidence of persistent circulation in poorer areas where access to clinical testing is limited. FUNDING: Bill & Melinda Gates Foundation.

Infection With Escherichia Coli Pathotypes Is Associated With Biomarkers of Gut Enteropathy and Nutritional Status Among Malnourished Children in Bangladesh.

Escherichia coli (E. coli) pathotypes are the most common cause of diarrhea, especially in developing countries. Environmental Enteric Dysfunction (EED) is presumed to be the result of infection with one or more pathotypes and can affect intestinal health and childhood growth. We sought to investigate the association of E. coli pathotypes infection with biomarkers of EED and nutritional status among slum-dwelling malnourished children in Bangladesh. This study comprised a total of 1050 stunted and at risk of stunting children. TaqMan Array Card assays were used to determine the presence of E. coli pathotypes in feces. Prevalence of infection with EAEC was highest (68.8%) in this cohort of children, followed by EPEC (55.9%), ETEC (44%), Shigella/EIEC (19.4%) and STEC (3.2%). The levels of myeloperoxidase and calprotectin were significantly higher in EAEC (P=0.02 and P=0.04), EPEC (P=0.02 and P=0.03) and Shigella/EIEC (P=0.05 and P=0.02) positive participants while, only calprotectin was significantly higher in ETEC (P=0.01) positive participants. Reg1B was significantly higher in participants with EAEC (P=0.004) while, neopterin levels were significantly lower in ETEC (P=0.003) and Shigella/EIEC (P=0.003) positive cases. A significant positive relationship was observed between EAEC and fecal levels of Reg1B (ß = 0.28; 95% CI = 0.12, 0.43; p-value<0.001). Besides, ETEC was found to be positively and significantly associated with the levels of calprotectin (ß = 0.14; 95 percent CI = 0.01, 0.26; p-value=0.037) and negatively with neopterin (ß = -0.16; 95% CI = -0.30, -0.02; p-value=0.021). On the other hand, infection with EPEC was found to be negatively associated with length-for-age (ß = -0.12; 95% CI = -0.22, -0.03; p-value=0.011) and weight-for-age (ß = -0.11; 95% CI = -0.22, -0.01; p-value=0.037). The study findings suggest that infection with certain E. coli pathotypes (EAEC and ETEC) influences gut health and EPEC is associated with linear growth and underweight in Bangladeshi children.

Small Intestine Bacterial Overgrowth in Bangladeshi Infants Is Associated With Growth Stunting in a Longitudinal Cohort.

INTRODUCTION: Small intestine bacterial overgrowth (SIBO) is common in children from low-income countries and has been cross-sectionally associated with growth stunting. We sought to determine whether SIBO was associated with poor growth and neurodevelopmental in a longitudinal analysis. METHODS: We measured SIBO by glucose hydrogen breath test (GHBT) at 18, 52, 78, and 104 weeks of life in a prospective longitudinal birth cohort of Bangladeshi children. Sociodemographic information and measures of enteric inflammation were analyzed as covariates. Diarrheal samples were tested for enteropathogens using polymerase chain reaction. Regression models were created using standardized mean GHBT area under the H2 curve (AUC) to determine associations with linear growth and cognitive, language, and motor scores on the Bayley-III Scales of Infant and Toddler Development at 2 years. We also investigated associations between GHBT AUC and enteropathogen exposure. RESULTS: A 1-ppm increase in standardized mean GHBT AUC was associated with a 0.01-SD decrease in length-for-age Z score (P = 0.03) and a 0.11-point decrease in Bayley language score (P = 0.05) at 2 years of age in adjusted analysis. Enteroaggregative Escherichia coli, Enteropathogenic Escherichia coli, Giardia, and Enterocytozoon bieneusi were associated with increased GHBT AUC, whereas Clostridium difficile, norovirus GI, sapovirus, rotavirus, and Cryptosporidium were associated with decreased GHBT AUC. None were consistent across all 4 time points. DISCUSSION: SIBO in the first 2 years of life is associated with growth stunting and decreased language ability in Bangladeshi infants and may represent a modifiable risk factor in poor growth and neurodevelopment in low-income countries.

Multiscale model for forecasting Sabin 2 vaccine virus household and community transmission.

Since the global withdrawal of Sabin 2 oral poliovirus vaccine (OPV) from routine immunization, the Global Polio Eradication Initiative (GPEI) has reported multiple circulating vaccine-derived poliovirus type 2 (cVDPV2) outbreaks. Here, we generated an agent-based, mechanistic model designed to assess OPV-related vaccine virus transmission risk in populations with heterogeneous immunity, demography, and social mixing patterns. To showcase the utility of our model, we present a simulation of mOPV2-related Sabin 2 transmission in rural Matlab, Bangladesh based on stool samples collected from infants and their household contacts during an mOPV2 clinical trial. Sabin 2 transmission following the mOPV2 clinical trial was replicated by specifying multiple, heterogeneous contact rates based on household and community membership. Once calibrated, the model generated Matlab-specific insights regarding poliovirus transmission following an accidental point importation or mass vaccination event. We also show that assuming homogeneous contact rates (mass action), as is common of poliovirus forecast models, does not accurately represent the clinical trial and risks overestimating forecasted poliovirus outbreak probability. Our study identifies household and community structure as an important source of transmission heterogeneity when assessing OPV-related transmission risk and provides a calibratable framework for expanding these analyses to other populations. Trial Registration: ClinicalTrials.gov This trial is registered with clinicaltrials.gov, NCT02477046.

Use of TaqMan Array Cards to investigate the aetiological agents of diarrhoea among young infants with severe acute malnutrition.

OBJECTIVE: Studies involving less sensitive conventional microscopy and culture-based approaches have identified distinct differences in diarrhoeal aetiology in childhood malnutrition. Our study involved the use of an advanced molecular biology technique, the TaqMan Array Cards (TAC), to elucidate the diarrhoeal aetiology among young infants with severe acute malnutrition (SAM). METHOD: A total of 113 faecal samples was collected from SAM infants, aged 2-6 months, upon admission to the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) with complications of diarrhoea and related comorbidities. We used TAC for the detection of 29 different diarrhoeal enteropathogens from a single faecal sample. For comparison, we also analysed 25 diarrhoeal samples from well-nourished infants of similar age. RESULTS: Higher odds of detection of all bacterial enteropathogens were associated with diarrhoea among SAM infants. In particular, the detection of Aeromonas sp (aOR: 25.7, p = 0.011), Campylobacter sp (aOR: 9.6, p < 0.01) and ETEC (aOR: 5.2, p = 0.022) was significantly associated with diarrhoea among SAM infants in comparison to well-nourished infants. 80% higher odds of detection of rotavirus and norovirus GII were associated with diarrhoea among well-nourished infants in comparison to SAM infants (aOR: 0.2, p < 0.05). CONCLUSION: Our study findings demonstrate a difference in diarrhoeal aetiology among SAM and well-nourished young infants, which may be useful in providing an evidence-based logic for possible revision of treatment guidelines for treatment of young diarrhoeal infants with SAM in the early management of the menace of antimicrobial resistance.

Subtractive proteomics approach to Unravel the druggable proteins of the emerging pathogen Waddlia chondrophila and drug repositioning on its MurB protein.

Waddlia chondrophila is an emerging pathogen that has been implicated in numerous unpropitious pregnancy events in humans and ruminants. Taking into account its association with abortigenic events, possible modes of transmission, and future risk, immediate clinical measures are required to prevent widespread damage caused by this organism and hence this study. Here, a subtractive proteomics approach was employed to identify druggable proteins of W. chondrophila. Considering the essential genes, antibiotic resistance proteins, and virulence factors, 676 unique important proteins were initially identified for this bacterium. Afterward, NCBI BLASTp performed against human proteome identified 223 proteins that were further pushed into KEGG Automatic Annotation Server (KAAS) for automatic annotation. Using the information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 14 Waddlia specific metabolic pathways were identified with respect to humans. Analyzing the data from KAAS and KEGG databases, forty-eight metabolic pathway-dependent, and seventy metabolic pathway independent proteins were identified. Standalone BLAST search against DrugBank FDA approved drug targets revealed eight proteins that are finally considered druggable proteins. Prediction of three-dimensional structures was done for the eight proteins through homology modeling and the Ramachandran plot model showed six models as a valid prediction. Finally, virtual screening against MurB protein was performed using FDA approved drugs to employ the drug repositioning strategy. Three drugs showed promising docking results that can be used for therapeutic purposes against W. chondrophila following the clinical validation of the study.

Diarrheal Pathogens Associated With Growth and Neurodevelopment.

BACKGROUND: Diarrheal pathogens have been associated with linear growth deficits. The effect of diarrheal pathogens on growth is likely due to inflammation, which also adversely affects neurodevelopment. We hypothesized that diarrheagenic pathogens would be negatively associated with both growth and neurodevelopment. METHODS: We conducted a longitudinal birth cohort study of 250 children with diarrheal surveillance and measured pathogen burden in diarrheal samples using quantitative polymerase chain reaction. Pathogen attributable fraction estimates of diarrhea over the first 2 years of life, corrected for socioeconomic variables, were used to predict both growth and scores on the Bayley-III Scales of Infant and Toddler Development. RESULTS: One hundred eighty children were analyzed for growth and 162 for neurodevelopmental outcomes. Rotavirus, Campylobacter, and Shigella were the leading causes of diarrhea in year 1 while Shigella, Campylobacter, and heat-stable toxin-producing enterotoxigenic Escherichia coli were the leading causes in year 2. Norovirus was the only pathogen associated with length-for-age z score at 24 months and was positively associated (regression coefficient [RC], 0.42 [95% confidence interval {CI}, .04 to .80]). Norovirus (RC, 2.46 [95% CI, .05 to 4.87]) was also positively associated with cognitive scores while sapovirus (RC, -2.64 [95% CI, -4.80 to -.48]) and typical enteropathogenic E. coli (RC, -4.14 [95% CI, -8.02 to -.27]) were inversely associated. No pathogens were associated with language or motor scores. Significant maternal, socioeconomic, and perinatal predictors were identified for both growth and neurodevelopment. CONCLUSIONS: Maternal, prenatal, and socioeconomic factors were common predictors of growth and neurodevelopment. Only a limited number of diarrheal pathogens were associated with these outcomes.

The Early Evolution of Oral Poliovirus Vaccine Is Shaped by Strong Positive Selection and Tight Transmission Bottlenecks.

The emergence of circulating vaccine-derived polioviruses through evolution of the oral polio vaccine (OPV) poses a significant obstacle to polio eradication. Understanding the early genetic changes that occur as OPV evolves and transmits is important for preventing future outbreaks. Here, we use deep sequencing to define the evolutionary trajectories of type 2 OPV in a vaccine trial. By sequencing 497 longitudinal stool samples from 271 OPV2 recipients and household contacts, we were able to examine the extent of convergent evolution in vaccinated individuals and the amount of viral diversity that is transmitted. In addition to rapid reversion of key attenuating mutations, we identify strong selection at 19 sites across the genome. We find that a tight transmission bottleneck limits the onward transmission of these early adaptive mutations. Our results highlight the distinct evolutionary dynamics of live attenuated virus vaccines and have important implications for the success of next-generation OPV.

Effect of water, sanitation, handwashing and nutrition interventions on enteropathogens in children 14 months old: a cluster-randomized controlled trial in rural Bangladesh.

BACKGROUND: We evaluated the impact of low-cost water, sanitation, handwashing (WSH) and child nutrition interventions on enteropathogen carriage in the WASH Benefits cluster-randomized controlled trial in rural Bangladesh. METHODS: We analyzed 1411 routine fecal samples from children 14±2 months old in the WSH (n = 369), nutrition counseling plus lipid-based nutrient supplement (n = 353), nutrition plus WSH (n = 360), and control (n = 329) arms for 34 enteropathogens using quantitative PCR. Outcomes included the number of co-occurring pathogens; cumulative quantity of four stunting-associated pathogens; and prevalence and quantity of individual pathogens. Masked analysis was by intention-to-treat. RESULTS: 326 (99.1%) control children had one or more enteropathogens detected (mean 3.8±1.8). Children receiving WSH interventions had lower prevalence and quantity of individual viruses than controls (prevalence difference for norovirus: -11% [95% confidence interval [CI], -5 to -17%]; sapovirus: -9% [95%CI, -3 to -15%]; and adenovirus 40/41: -9% [95%CI, -2 to - 15%]). There was no difference in bacteria, parasites, or cumulative quantity of stunting-associated pathogens between controls and any intervention arm. CONCLUSIONS: WSH interventions were associated with fewer enteric viruses in children aged 14 months. Different strategies are needed to reduce enteric bacteria and parasites at this critical young age.

A Highly Conserved GEQYQQLR Epitope Has Been Identified in the Nucleoprotein of Ebola Virus by Using an In Silico Approach.

Ebola virus (EBOV) is a deadly virus that has caused several fatal outbreaks. Recently it caused another outbreak and resulted in thousands afflicted cases. Effective and approved vaccine or therapeutic treatment against this virus is still absent. In this study, we aimed to predict B-cell epitopes from several EBOV encoded proteins which may aid in developing new antibody-based therapeutics or viral antigen detection method against this virus. Multiple sequence alignment (MSA) was performed for the identification of conserved region among glycoprotein (GP), nucleoprotein (NP), and viral structural proteins (VP40, VP35, and VP24) of EBOV. Next, different consensus immunogenic and conserved sites were predicted from the conserved region(s) using various computational tools which are available in Immune Epitope Database (IEDB). Among GP, NP, VP40, VP35, and VP30 protein, only NP gave a 100% conserved GEQYQQLR B-cell epitope that fulfills the ideal features of an effective B-cell epitope and could lead a way in the milieu of Ebola treatment. However, successful in vivo and in vitro studies are prerequisite to determine the actual potency of our predicted epitope and establishing it as a preventing medication against all the fatal strains of EBOV.

Structural, functional and molecular docking study to characterize GMI1 from Arabidopsis thaliana.

γ-irradiation and Mitomycin C Induced 1 (GMI1), is a member of the SMC-hinge domain-containing protein family that takes part in double stranded break repair mechanism in eukaryotic cells. In this study we hypothesize a small molecule-Adenosine Tri Phosphate (ATP) binding region of novel SMC like GM1 protein in model organism Arabidopsis thaliana using in silico modeling. Initially, analyzing sequence information for the protein indicated presence of motifs - 'Walker A nucleotide-binding domain' that are required to interact with nucleotides along with 'Walker B' motif and ABC signature sequences. This was further proven through GMI1-ATP docking experiment and results were verified by comparing the values with controls. In negative control, no binding was seen in the same binding region of GMI1 structure for small molecules randomly selected form PubChem database, whereas in positive control binding affinity of other known proteins with ATP binding potential resembled GMI1-ATP binding affinity of -5.4 kcal/mol. Furthermore we also docked small molecules that shares structural similarity with ATP to GMI1 and found that Purine Mononucleotide bound the region with the best affinity, which implies that the compound may bind the protein with strong binding and can work as a potential agonist/antagonist to GMI1. We believe that the study would shed more light into the GM1 mechanism of action. Although the computational predictions made here are based on concrete confidence, it should be mentioned that in vitro experimentation does not fall into the scopes of this study and thus the results found here have to be further validated in vitro.