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Natural radioactivity has been determined by means of a highly efficient thallium-doped sodium iodide detector in soil sample from surface of from vicinage of "Kolaghat Thermal Power Plant" The activity concentration (specific activity) is found to vary from 23.70 ± 3.41 to 33.90 ± 4.33 Bqkg-1 for 226Ra, from 6.67 ± 2.01 to 21.60 ± 2.11 Bqkg-1 for 232Th and that for 40K from 461.01 ± 66.16 to 610.25 ± 80.87 Bqkg-1 with expected value of 29.26 ± 3.50, 4.86 ± 2.03 and 517.53 ± 71.61 Bqkg-1 of Radium-226, Thorium-232 and Potassium-40 correspondingly. The radon exhalation rates were also computed from all samples using the "Sealed Can Technique". The activity due to radon fluctuates from 47.9 ± 6.2 to 157.6 ± 11.3 Bqm-3 having an arithmetic mean of 89.3 ± 7.9 Bqm-3. The surface exhalation rate and mass exhalation rates were estimated to fluctuate from17.3 ± 2.2 to 56.8 ± 4.1 mBqm-2h-1 with an expectation of 32.1 ± 3.0 mBqm-2h-1 and from 0.6 ± 0.1 to 2.1 ± 0.2 mBqkg-1h-1 averaged out to 1.2 ± 0.1 mBqkg-1h-1respectively. Radiation doses were also calculated in these samples. Indoor and outdoor annual effective doses, Absorbed dose rates, External Hazard index and Indoor inhalation exposure, also known as radon effective dose, was computed and varied from 0.19 to 0.25 mSv y-1, 39.30 to 51.61 nGyh-1, 0.048 to 0.063 mSvy-1, 0.21 to 0.28 and 0.29 to 0.36 and 2.04 to 6.70 µSvy-1 respectively. The values which were obtained in this study are found to be well below the allowed permissible limits, so the soil may be used as building construction material. [ABSTRACT FROM AUTHOR]

Background: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers. Objective: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth. Design/Methods: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman’s correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration. Results: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, β (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA. Conclusions and relevance: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions. [ABSTRACT FROM AUTHOR]

Enteropathies such as Crohn’s disease are associated with enteric inflammation characterized by impaired TGF-β signaling, decreased expression of phosphorylated (p)-SMAD2,3 and increased expression of SMAD7 (an inhibitor of SMAD3 phosphorylation). Environmental enteropathy (EE) is an acquired inflammatory disease of the small intestine (SI), which is associated with linear growth disruption, cognitive deficits, and reduced oral vaccine responsiveness in children <5 y in resource-poor countries. We aimed to characterize EE inflammatory pathways by determining SMAD7 and p-SMAD2,3 levels (using Western blotting) in EE duodenal biopsies (N = 19 children, 7 from Pakistan, 12 from Zambia) and comparing these with healthy controls (Ctl) and celiac disease (CD) patients from Italy. Densitometric analysis of immunoblots showed that EE SI biopsies expressed higher levels of both SMAD7 (mean±SD in arbitrary units [a.u.], Ctl = 0.47±0.20 a.u., EE = 1.13±0.25 a.u., p-value = 0.03) and p-SMAD2,3 (mean±SD, Ctl = 0.38±0.14 a.u., EE = 0.60±0.10 a.u., p-value = 0.03). immunohistochemistry showed that, in EE, SMAD7 is expressed in both the epithelium and in mononuclear cells of the lamina propria (LP). In contrast, p-SMAD3 in EE is expressed much more prominently in epithelial cells than in the LP. The high SMAD7 immunoreactivity and lack of p-SMAD2,3 expression in the LP suggests defective TGF-β signaling in the LP in EE similar to a previously reported SMAD7-mediated inflammatory pathway in refractory CD and Crohn’s disease. However, Western blot densitometry showed elevated p-SMAD2,3 levels in EE, possibly suggesting a different inflammatory pathway than Crohn’s disease but more likely reflecting cumulative protein expression from across all compartments of the mucosa as opposed to the LP alone. Further studies are needed to substantiate these preliminary results and to illustrate the relationship between SMAD proteins, TGF-β signaling, and inflammatory cytokine production, all of which may be potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Acute respiratory infections (ARI) play a major role in hospitalizations in the Middle East, but the specific viral causes are unknown. We conducted prospective viral surveillance in children <5 y of age admitted with ARI and/or fever at 2 dissimilar hospitals in Amman, Jordan during peak respiratory syncytial virus (RSV) season. We collected prospective clinical and demographic data and obtained nose/throat swabs for testing for RSV by real-time polymerase chain reaction (RT-PCR). We obtained clinical and laboratory data for 728/743 (98%) subjects enrolled. The children's median age was 4.3 months, 58.4% were males, 87% were breastfed, 4% attended day care, 67% were exposed to smokers, 7% were admitted to the intensive care unit, and 0.7% died ( n = 5). Out of 728 subjects, 467 (64%) tested positive by RT-PCR for RSV. Comparing RSV-positive with RSV-negative subjects, the RSV-positive subjects had lower median age (3.6 vs 6.4 months, p < 0.001) and fewer males (55% vs 64%, p = 0.02). RSV-positive children had higher rates of oxygen use (72% vs 42%, p < 0.001), a longer hospital stay (5 vs 4 days, p = 0.001), and higher hospital charges (US$538 vs US$431, p < 0.001) than RSV-negative children. In young hospitalized Jordanian infants, the medical and financial burden of RSV was found to be high. Effective preventive measures, such as an RSV vaccine, would have a significant beneficial impact. [ABSTRACT FROM AUTHOR]

In the present study, we estimate the effect of paints on the radon exhalation rate from building materials. For the experimental study some bricks were collected from the local market of Aligarh district, Uttar Pradesh, India. They were coated with a soup of cement and sand, and then dried for at least 24 h. After drying, the coated bricks were plastered with whitewash and re-dried for a minimum of 2 h. They were then ready to be painted with different colours of paints from different brands. Radon exhalation rate and effective dose were measured for the painted brick samples by adopting sealed can technique, which utilizes LR-115 type-II track detectors. The radon activity varied from 768.8 to 1529.8 Bq m-3 and exhalation rate varied from 460.1 to 915.6 mBq m-2 h-1, whereas the dose equivalent for radon daughters varied from 54.2 to 107 µSv yr-1. Exhalation of radon in the case of Snowcem (limeproof yellow), Asian Paints (peach organza) and Berger Paints (yellow breeze) was found to increase slightly, while it decreased for the other paint brands. [ABSTRACT FROM AUTHOR]

Ready-to-use supplemental foods (RUSF) are energy-dense meals formulated to prevent and treat moderate and severe childhood acute malnutrition (MAM and SAM) in high-risk settings. Although lifesaving, the degree and durability of weight recovery with RUSF is unpredictable. We examined whether environmental enteric dysfunction (EED) and gut microbiota perturbations are risk factors for RUSF failure in a birth cohort of 416 rural Pakistani children followed for growth, common childhood illnesses, and biomarkers from blood, urine, and stool. Infants who developed wasting (weight-for-length Z score <-2, n=187, 45%) during surveillance received Acha Mum (a chickpea-based RUSF) daily for eight weeks. Machine learning identified seven biomarkers that predicted RUSF response (n=75) vs. non-response (n=112) with 73% accuracy. Remarkably, gut microbiome composition predicted RUSF response with 93% (pre-supplementation) and 98% (post-supplementation) accuracy. Seven outliers whose microbiome falsely predicted positive response experienced extraordinary burdens of inflammation and illness during supplementation. These findings identify gut microbial signatures and biomarkers of gut and systemic inflammation as robust predictors of RUSF response in infants free from intercurrent illness during recovery, setting the stage for predictive models to guide precision use of RUSF and adjunct therapies in undernourished children.

Background: Environmental enteric dysfunction (EED) is an inflammatory condition of the small intestine that is prevalent in children residing in low- and middle-income countries. EED is accompanied by profound histopathologic changes in the small bowel, loss of absorptive capacity, increased intestinal permeability, increased microbial translocation, and nutrient loss., Objectives: We sought to identify dysregulated genes and pathways that might underlie pediatric EED., Methods: RNA-sequencing libraries were generated from endoscopically obtained duodenal tissue from undernourished children with EED from 3 prospective cohorts of children with EED. The EED transcriptome was defined in comparison to North American children without EED. Weighted gene coexpression network analysis (WGCNA) was tested for gene modules associated with EED and its histologic features., Results: The 1784 upregulated genes in EED were highly enriched for immune and inflammatory processes, including IL-17 and JAK-STAT signaling, and cytokine-cytokine receptor interactions. The 1388 downregulated genes included genes corresponding to xenobiotic metabolism, detoxification, and antioxidant capacities. A gene coexpression module enriched for antimicrobial responses and chemokine activity was significantly associated with villous blunting, goblet cell depletion, and overall histologic severity of EED., Conclusions: The transcriptome signatures of EED include specific innate and adaptive immune responses that are consistently elevated across study centers, coupled with reduced detoxification and antioxidant capacities. These data may have implications for targeted interventions to improve EED outcomes., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Environmental enteric dysfunction (EED) is characterized by reduced absorptive capacity and barrier function of the small intestine, leading to poor ponderal and linear childhood growth., Objectives: To further define gene expression patterns that are associated with EED to uncover new pathophysiology of this disorder., Methods: Duodenal biopsies from cohorts of children with EED from Bangladesh, Pakistan and Zambia were analyzed by immunohistochemistry (IHC) to interrogate gene products that distinguished differentiation and various biochemical pathways in immune and epithelial cells, some identified by prior bulk RNA sequence analyses. Immunohistochemical staining was digitally quantified from scanned images and compared to cohorts of North American children with celiac disease (gluten-sensitive enteropathy) or with no known enteric disease and no pathologic abnormality (NPA) detected in their clinical biopsies., Results: After multivariable statistical analysis, we identified statistically significant (P < 0.05, 2-tailed t-test) elevated signals representing cluster of differentiation 45 (80%; 95% confidence interval [CI]: 24%, 127%), lipocalin 2 (659%; 95% CI: 198%, 1838%), and regenerating family 1 beta (221%; 95% CI: 47%, 600%) and lower signals corresponding to granzyme B (-74%; 95% CI: -82%, -62%), and sucrase isomaltase (-58%; 95% CI: -75%, -29%) in EED biopsies compared with NPA biopsies. Computerized algorithms also detected statistically significant elevation in intraepithelial lymphocytes (49%; 95% CI: 9%, 105%) and proliferation of leukocytes (267%; 95% CI: 92%, 601%) in EED biopsies compared with NPA biopsies., Conclusions: Our results support a model of chronic epithelial stress that decreases epithelial differentiation and absorptive function. The close association of several IHC parameters with manual histologic scoring suggests that automated digital quantification of IHC panels complements traditional histomorphologic assessment in EED., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Environmental enteric dysfunction (EED) is an asymptomatic acquired disorder characterized by upper small bowel inflammation, villus blunting, and gut permeability. It is a major contributor to poor growth in childhood as well as other highly consequential outcomes such as delayed neuorcognitive development. After decades of intermittent interest in this entity, we are now seeing a resurgence in the field of EED. However, recent studies have been hampered by a lack of investigation of the target tissue-the upper small bowel. In 2016, the EEDBI (Environmental Enteric Dysfunction Biopsy Initiative) Consortium was established as a common scientific platform across 3 independent EED biopsy cohort studies in Bangladesh, Pakistan, and Zambia. Two centers in the United States recruited comparison groups of children undergoing endoscopy for clinical indications. The EEDBI Consortium goal was to augment the contributions of the individual centers and answer high-level questions amenable to analysis and interpretation across the studies. Here, we describe the Consortium and its cohorts and recruitment procedures across studies. We also offer details applicable to all papers in this supplement, which describe EED mucosal histology, morphometry, immunohistochemistry, and transcriptomics as well as histology relationship to pathogens and biomarkers., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Environmental enteric dysfunction (EED) is a precursor of growth faltering in children living in impoverished conditions who are frequently exposed to environmental toxins and enteropathogens, leading to small bowel inflammatory, malabsorptive, and permeability derangements and low-grade chronic systemic inflammation., Objectives: We explored the association between anthropometrics and duodenal histologic features of EED among children from 3 lower middle-income country centers., Methods: In this cross-sectional study, Pakistani children (n = 63) with wasting, Bangladesh children (n = 116) with stunting or at risk for stunting (height-for-age Z score [HAZ] <-1 but ≥-2), and Zambian children (n = 108) with wasting or stunting received nutritional intervention. Children with anthropometric status refractory to intervention underwent endoscopy. Linear regression models included anthropometric around endoscopy, scores of histology parameters, and a global index score of EED-the total score percent-5 (TSP-5). Multivariable models were adjusted for center, age, sex, and histology slide quality., Results: Intersite variation was observed while exploring the association between anthropometrics and the TSP-5; for example, Pakistani children had the worst HAZ, yet their median TSP-5 score was lower than that of the other 2 centers. Even within each site, no overall pattern of higher TSP-5 score was observed with worsening HAZ. During univariate analysis, TSP-5 (coefficient: 0.01; 95% confidence interval [CI]: 0, 0.02), goblet cell depletion (coefficient: 0.22; 95% CI: 0.06, 0.37), and Paneth cell depletion (coefficient: 0.14; 95% CI: 0.01, 0.27) were associated with HAZ scores; however, they lost statistical significance in the multivariable models, with study center most strongly confounding the relationships seen in univariate models between anthropometry and histology., Conclusions: This study contributes a crucial negative finding that duodenal morphological features did not associate with anthropometric phenotypes; hence, anthropometric measurements may not be a suitable outcome measure for use in EED trials. Trial outcomes may need to be defined by combining the functional and structural elements of the gut to monitor EED., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Validated biomarkers could catalyze environmental enteric dysfunction (EED) research., Objectives: Leveraging an EED histology scoring system, this multicountry analysis examined biomarker associations with duodenal histology features among children with EED. We also examined differences in 2-h compared with 1-h urine collections in the lactulose rhamnose (LR) dual sugar test., Methods: Three cohorts of undernourished children unresponsive to nutrition intervention underwent esophagogastroduodenoscopy and duodenal biopsies. Histopathology scores were compared to fecal calprotectin (CAL), myeloperoxidase (MPO), neopterin (NEO), and urinary LR ratio and lactulose percentage recovery. Log-transformed biomarkers were used in linear regressions adjusted for age, center, and sample collection-biopsy time interval in multivariable models., Results: Data on >1 biomarker were available for 120 Bangladeshi (CAL, MPO, NEO, and LR), 63 Pakistani (MPO, NEO, and LR), and 63 Zambian children (CAL). Median age at endoscopy was similar (19 mo) across centers. Median sample collection prior to endoscopy was consistent with each center's study design: 2 wk in Bangladesh (urine and stool) and Zambia (stool), and 6 (urine) and 11 (stool) mo in Pakistan. In multivariable models, intraepithelial lymphocytes were associated with CAL (exponentiated [exp.] coefficient: 1.19; 95% confidence interval [CI]: 1, 1.41), intramucosal Brunner's glands with MPO (exp. coefficient: 1.33; 95% CI: 1.05, 1.69) and NEO (exp. coefficient: 1.37; 95% CI: 1.1, 1.7), and chronic inflammation with NEO (exp. coefficient: 1.61; 95% CI: 1.17, 2.17). Intraepithelial lymphocytes were associated with lactulose % recovery (exp. coefficient: 1.22; 95% CI: 1.05, 1.41). LR recovery was substantially lower in 1-h collections than in 2-h collections., Conclusions: Four commonly used markers of enteric dysfunction were associated with specific histologic features. One-hour urine collection may be insufficient to reflect small bowel permeability in LR testing. While acknowledging the challenges with obtaining relevant tissue, these findings form the basis for further EED biomarker validation research., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Environmental enteric dysfunction (EED) is an asymptomatic intestinal disorder associated with growth impairment, delayed neurocognitive development, and impaired oral vaccine responses., Objectives: We set out to develop and validate a histopathologic scoring system on duodenal biopsies from a cohort study of children with growth failure in Bangladesh, Pakistan, and Zambia ("EED") with reference to biopsies from United States children with no clinically reported histologic pathology (referred to hereafter as "normal") or celiac disease., Methods: Five gastrointestinal pathologists evaluated 745 hematoxylin and eosin slide images from 291 children with EED (mean age: 1.6 y) and 66 United States children (mean age: 6.8 y). Histomorphologic features (i.e., villus/crypt architecture, goblet cells, epithelial and lamina propria acute/chronic inflammation, Brunner's glands, Paneth cells, epithelial detachment, enterocyte injury, and foveolar metaplasia) were used to score each histopathologic slide. Generalized estimating equations were used to determine differences between EED, normal, and celiac disease, and receiver operating characteristic curves were used to assess predictive value., Results: Biopsies from the duodenal bulb showed higher intramucosal Brunner's gland scores and lower intraepithelial lymphocyte scores than from the second or third parts of the duodenum (D2/3), so only D2/3 were included in the final analysis. Although 7 parameters differed significantly between EED and normal biopsies in regression models, only 5 (blunted villus architecture, increased intraepithelial lymphocytosis, goblet cell depletion, Paneth cell depletion, and reduced intramucosal Brunner's glands) were required to create a total score percentage (TSP-5) that correctly identified EED against normal biopsies (AUC: 0.992; 95% CI: 0.983, 0.998). Geographic comparisons showed more severe goblet cell depletion in Bangladesh and more marked intraepithelial lymphocytosis in Pakistan., Conclusions: This scoring system involving 5 histologic parameters demonstrates very high discrimination between EED and normal biopsies, indicating that this scoring system can be applied with confidence to studies of intestinal biopsies in EED., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Environmental enteric dysfunction (EED), a chronic inflammatory condition of the small intestine, is an important driver of childhood malnutrition globally. Quantifying intestinal morphology in EED allows for exploration of its association with functional and disease outcomes., Objectives: We sought to define morphometric characteristics of childhood EED and determine whether morphology features were associated with disease pathophysiology., Methods: Morphometric measurements and histology were assessed on duodenal biopsy slides for this cross-sectional study from children with EED in Bangladesh, Pakistan, and Zambia (n = 69), and those with no pathologic abnormality (NPA; n = 8) or celiac disease (n = 18) in North America. Immunohistochemistry was also conducted on 46, 8, and 18 biopsy slides, respectively. Linear mixed-effects regression models were used to reveal morphometric differences between EED compared with NPA or celiac disease and identify associations between morphometry and histology or immunohistochemistry among children with EED., Results: In duodenal biopsies, median EED villus height (248 μm), crypt depth (299 μm), and villus:crypt (V:C) ratio (0.9) values ranged between those of NPA (396 μm villus height; 246 μm crypt depth; 1.6 V:C ratio) and celiac disease (208 μm villus height; 365 μm crypt depth; 0.5 V:C ratio). Among EED biopsy slides, morphometric assessments were not associated with histologic parameters or immunohistochemical markers, other than pathologist-determined subjective semiquantitative villus architecture., Conclusions: Morphometric analysis of duodenal biopsy slides across geographies identified morphologic features of EED, specifically short villi, elongated crypts, and a smaller V:C ratio relative to NPA slides, although not as severe as in celiac slides. Morphometry did not explain other EED features, suggesting that EED histopathologic processes may be operating independently of morphology. Although acknowledging the challenges with obtaining relevant tissue, these data form the basis for further assessments of the role of morphometry in EED., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hematoxylin and Eosin (H&E) stained Whole Slide Images (WSIs) are utilized for biopsy visualization-based diagnostic and prognostic assessment of diseases. Variation in the H&E staining process across different lab sites can lead to significant variations in biopsy image appearance. These variations introduce an undesirable bias when the slides are examined by pathologists or used for training deep learning models. Traditionally proposed stain normalization and color augmentation strategies can handle the human level bias. But deep learning models can easily disentangle the linear transformation used in these approaches, resulting in undesirable bias and lack of generalization. To handle these limitations, we propose a Self-Attentive Adversarial Stain Normalization (SAASN) approach for the normalization of multiple stain appearances to a common domain. This unsupervised generative adversarial approach includes self-attention mechanism for synthesizing images with finer detail while preserving the structural consistency of the biopsy features during translation. SAASN demonstrates consistent and superior performance compared to other popular stain normalization techniques on H&E stained duodenal biopsy image data.

Celiac Disease (CD) and Environmental Enteropathy (EE) are common causes of malnutrition and adversely impact normal childhood development. CD is an autoimmune disorder that is prevalent worldwide and is caused by an increased sensitivity to gluten. Gluten exposure destructs the small intestinal epithelial barrier, resulting in nutrient mal-absorption and childhood under-nutrition. EE also results in barrier dysfunction but is thought to be caused by an increased vulnerability to infections. EE has been implicated as the predominant cause of under-nutrition, oral vaccine failure, and impaired cognitive development in low-and-middle-income countries. Both conditions require a tissue biopsy for diagnosis, and a major challenge of interpreting clinical biopsy images to differentiate between these gastrointestinal diseases is striking histopathologic overlap between them. In the current study, we propose a convolutional neural network (CNN) to classify duodenal biopsy images from subjects with CD, EE, and healthy controls. We evaluated the performance of our proposed model using a large cohort containing 1000 biopsy images. Our evaluations show that the proposed model achieves an area under ROC of 0.99, 1.00, and 0.97 for CD, EE, and healthy controls, respectively. These results demonstrate the discriminative power of the proposed model in duodenal biopsies classification.

Background: Molecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. Molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (DFA) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. We sought to perform a real-world comparison of two molecular respiratory viral diagnostic methods between two experienced respiratory virus research laboratories., Methods: We tested nasal and throat swab specimens obtained from 225 infants with respiratory illness for 11 common respiratory viruses using both a multiplex assay (Respiratory MultiCode-PLx Assay [RMA]) and individual real-time RT-PCR (RT-rtPCR)., Results: Both assays detected viruses in more than 70% of specimens, but there was discordance. The RMA assay detected significantly more human metapneumovirus (HMPV) and respiratory syncytial virus (RSV), while RT-rtPCR detected significantly more influenza A. We speculated that primer differences accounted for these discrepancies and redesigned the primers and probes for influenza A in the RMA assay, and for HMPV and RSV in the RT-rtPCR assay. The tests were then repeated and again compared. The new primers led to improved detection of HMPV and RSV by RT-rtPCR assay, but the RMA assay remained similar in terms of influenza detection., Conclusions: Given the absence of a gold standard, clinical and research laboratories should regularly correlate the results of molecular assays with other PCR based assays, other laboratories, and with standard virologic methods to ensure consistency and accuracy.