Novel, Rapid and Cost-effective Methods for Concentration, Detection and Monitoring of Waterborne Pathogens in Resource-Limited Settings

Waterborne pathogenic organisms including bacteria, viruses, protozoa and helminths, are responsible for a series of diseases which is a major public health concern worldwide. This issue is extremely severe in developing regions due to the scarcity of clean water supply and poor sanitation. Therefore, point-of-use (POU) detection and quantification processes as well as a monitoring program of waterborne pathogens are needed to ensure the safety of water and protect human health. However, the polymerase chain reaction (PCR) technology and its related detection platforms rely on complicated thermal cycling, centralized laboratory equipment and trained personnel, thus making PCR-based systems incapable of POU testing of environmental waters. In this dissertation, we develop a portable 3D-printed system with super-absorbent polymer (SAP) microspheres for sample enrichment, and a membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system for absolute quantification of pathogens. We also explored the interactions between microbial indicator of Escherichia coli (E. coli) and waterborne pathogen Vibrio Cholerae (V. Cholerae). The main results are as follows: 1. The application of detection and quantification methods is often hindered by the low pathogen concentrations in natural waters. Rapid and efficient sample concentration methods are urgently needed. Here we present a novel method to pre-concentrate microbial pathogens in water using a portable 3D-printed system with super-absorbent polymer (SAP) microspheres, which can effectively reduce the actual volume of water in a collected sample. The SAP microspheres absorb water while excluding bacteria and viruses by size exclusion and charge repulsion. The 3D-printed system with optimally-designed SAP microspheres could rapidly achieve a 10-fold increase in the concentration of E. coli and bacteriophage MS2 within 20 minutes with concentration efficiencies of 87% and 96%, respectively. Fold changes between concentrated and original samples from qPCR and RT-qPCR results were found to be 11.34-22.27 for E. coli with original concentrations of 104-106 cell·mL-1; and 8.20-13.81 for MS2 with original concentrations of 104-106 PFU·mL-1. Furthermore, SAP microspheres can be reused 20 times without performance loss thereby significantly decreasing the cost of our concentration system. 2. Following sample concentration, accurate quantification methods for waterborne pathogens are needed, especially at the point of sample collection. The surge of COVID-19 in late 2019 called for a more urgent need for a rapid and cost-effective quantification of SARS-CoV-2 in environmental waters. Quantification results contribute to wastewater-based epidemiology (WBE) which helps the monitoring of prevalent infections within a community and early detections of contamination. Here we demonstrated the usage of our portable membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system for absolute quantification of SARS CoV-2 in wastewater samples within a one-hour timeframe for point-of-use (POU) testing and data management. The limit of detection (LOD) of mgLAMP for SARS-CoV-2 quantification in Milli-Q water was observed to be down to 1 copy/mL, and that in surface water collected from Kathmandu, Nepal was down to 100 copies/mL. Both were 100-fold lower than that of RT-qPCR in corresponding matrices. Compared to alternative detection methods, our platform has a very high level of tolerance against inhibitors thanks to the restriction of the hydrogel matrix. This enables the highly sensitive detection in either clinical or environmental samples. 3. Regular environmental surveillance of waterborne pathogens is key to ensure the safety of water and protect public health. Due to the diversity of pathogenic bacteria in environmental waters, regular monitoring of so many pathogens for individuality is impractical. Therefore, microbial indicators are used to gauge the total pathogen concentration; and manage waterborne health risks. In this study, the interactions of V. cholerae, the etiologic agent of reemerging cholera, with E. coli, the most commonly used indicator for waterborne pathogens. Specifically, we investigated through evaluating the survival and growth of both bacteria under different temperature and nutrition deprivation using plate culturing and real-time polymerase chain reaction (qPCR). During co-growth, it was challenging for V. Cholerae to maintain initial population advantages as E. coli consumes nutrition more effectively. Whereas during co-existence, V. Cholerae soon fell into a viable-but–non-culturable state under environmental stress in 3-5 days while E. coli stay viable more than 14 days. We found that V. cholerae interacts with E. coli differently depending on the composition of the water that is sampled and analyzed. This suggests that bacterium-bacterium interactions influenced by the intrinsic chemical and biological parameters of ambient water will be a contributing mechanism in regulating the proliferation of V. cholerae. In summary, two platforms for environmental sample concentration and detection have been developed and tested using ambient and engineered waters. In addition, interactions between a microbial indicator, E. coli, and the pathogenic bacteria, V. Cholerae, were studied. The chapters in this thesis describe in detail: (1) A hand-pressed 3D-printed system to produce SAP microspheres was developed with the goal of achieving efficient concentrations of environmental microorganisms for subsequent analysis. The simplified concentration procedure and can be easily integrated into various detection platforms; (2) A portable membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system was developed for absolute quantification of SARS-CoV-2 in environmental water samples within one hour, enabling a 100-fold lower detection limit compared to the gold-standard of RT-qPCR; and (3) Differences in bacterium-bacterium interactions of V. cholerae and E. coli under as a function of water composition indicated that environmental stress presented in ambient water matrices should be taken into consideration while using a microbial indicator such as E. coli to estimate the risk of waterborne pathogens. These collective advances allow for the rapid and ultrasensitive POU testing of waterborne pathogens that should provide for more effective monitoring strategies in terms of the use of indicator microorganisms.</p

Synthesis and Application of Superabsorbent Polymer Microspheres for Rapid Concentration and Quantification of Microbial Pathogens in Ambient Water

Even though numerous methods have been developed for the detection and quantification of waterborne pathogens, the application of these methods is often hindered by the very low pathogen concentrations in natural waters. Therefore, rapid and efficient sample concentration methods are urgently needed. Here we present a novel method to pre-concentrate microbial pathogens in water using a portable 3D-printed system with super-absorbent polymer (SAP) microspheres, which can effectively reduce the actual volume of water in a collected sample. The SAP microspheres absorb water while excluding bacteria and viruses by size exclusion and charge repulsion. To improve the water absorption capacity of SAP in varying ionic strength waters (0-100 mM), we optimized the formulation of SAP to 180 g∙L⁻¹ Acrylamide, 75 g∙L⁻¹ Itaconic Acid and 4.0 g∙L⁻¹ Bis-Acrylamide for the highest ionic strength water as a function of the extent of cross-linking and the concentration of counter ions. Fluorescence microscopy and double-layer agar plating respectively showed that the 3D-printed system with optimally-designed SAP microspheres could rapidly achieve a 10-fold increase in the concentration of Escherichia coli (E. coli) and bacteriophage MS2 within 20 minutes with concentration efficiencies of 87% and 96%, respectively. Fold changes between concentrated and original samples from qPCR and RT-qPCR results were found to be respectively 11.34-22.27 for E. coli with original concentrations from 10⁴ to 10⁶ cell·mL⁻¹, and 8.20-13.81 for MS2 with original concentrations from 10⁴-10⁶ PFU·mL⁻¹. Furthermore, SAP microspheres can be reused for 20 times without performance loss, significantly decreasing the cost of our concentration system

Synthesis and Application of Superabsorbent Polymer Microspheres for Rapid Concentration and Quantification of Microbial Pathogens in Ambient Water

Even though numerous methods have been developed for the detection and quantification of waterborne pathogens, the application of these methods is often hindered by the very low pathogen concentrations in natural waters. Therefore, rapid and efficient sample concentration methods are urgently needed. Here we present a novel method to pre-concentrate microbial pathogens in water using a portable 3D-printed system with super-absorbent polymer (SAP) microspheres, which can effectively reduce the actual volume of water in a collected sample. The SAP microspheres absorb water while excluding bacteria and viruses by size exclusion and charge repulsion. To improve the water absorption capacity of SAP in varying ionic strength waters (0-100 mM), we optimized the formulation of SAP to 180 g∙L⁻¹ Acrylamide, 75 g∙L⁻¹ Itaconic Acid and 4.0 g∙L⁻¹ Bis-Acrylamide for the highest ionic strength water as a function of the extent of cross-linking and the concentration of counter ions. Fluorescence microscopy and double-layer agar plating respectively showed that the 3D-printed system with optimally-designed SAP microspheres could rapidly achieve a 10-fold increase in the concentration of Escherichia coli (E. coli) and bacteriophage MS2 within 20 minutes with concentration efficiencies of 87% and 96%, respectively. Fold changes between concentrated and original samples from qPCR and RT-qPCR results were found to be respectively 11.34-22.27 for E. coli with original concentrations from 10⁴ to 10⁶ cell·mL⁻¹, and 8.20-13.81 for MS2 with original concentrations from 10⁴-10⁶ PFU·mL⁻¹. Furthermore, SAP microspheres can be reused for 20 times without performance loss, significantly decreasing the cost of our concentration system

Nomogram for Predicting Bone Development State of Female Children and Adolescents–A Fast Screening Approach Based on Pubes Stages for Growth and Development

Objective: To develop a nomogram for predicting bone development state (BDS) of female children and adolescents in a large scale.Methods: Four hundred forty-seven female students were designated as the training cohort to develop the predictive model, whereas 196 female students were used as the validation cohort to verify the established model. Bone age, height, body mass, body fat percentage, and secondary sexual characteristics were recorded, and BDS was determined with the chronological age and bone age. Multivariate logistic regression was conducted to determine the factors, and nomogram was developed and validated with the training and validation cohorts, respectively.Results: One hundred forty-seven female students were identified as BDS abnormal in the training cohort (32.9%), and 104 were determined in the validation cohort (53.1%). Age, height, weight, and pubes stage were selected for the predictive model. A nomogram was developed and showed a good estimation, with a C-index of 0.78 and a good calibration in the training cohort. Application of the nomogram to the validation cohort showed a similar C-index of 0.75 and a good calibration.Conclusion: A nomogram for predicting bone development was developed, which can provide a relatively good estimation of BDS for female children and adolescents in Chinese metropolis

Long-term treatment with Perampanel of Chinese patients with focal-onset seizures, especially in sleep-related epilepsy: a prospective real-world observational study

BackgroundThere is currently a lack of studies examining the long-term therapeutic effectiveness of the third-generation anti-sezure medication, perampanel (PER), for focal-onset seizures (FOS), particularly in Chinese patients with sleep-related epilepsy (SRE). Additionally, the appropriate dosage, plasma concentration, and the relationship between dose and plasma concentration of PER in Chinese patients are still uncertain.MethodsA prospective, single-center, 24-month observational study was conducted in patients diagnosed with FOS, with a focus on patients with SRE. Changes in seizure frequency from baseline, adverse events, and retention rates were analyzed at 12 and 24 months following the start of the treatment. Tolerability was evaluated based on adverse events and discontinuation profiles. PER plasma concentrations were used to assess dose-concentration-response relationships.ResultsA total of 175 patients were included (median age: 25 years; range: 4–72 years; 53. 1% males and 46.9% females), with the SRE population accounting for 49. 1% (n = 86). The patients diagnosed with SRE showed considerably higher response rates than those who did not have this diagnosis (p = 0.025, odds ratio = 3.8). Additionally, the SRE group adhered better to PER treatment (r = 0.0009). Patients with a shorter duration of epilepsy (median: 3 years; range:2–7 years) demonstrated a more favorable therapeutic response to PER (p = 0.032). Throughout the administration of maintenance doses, among the entire FOS population, the concentration of PER (C0) ranged between 101.5 and 917.4 ng/mL (median, 232.0 ng/mL), and the mean plasma concentration of PER in the responders was 292.8 ng/mL. We revealed a linear relationship between PER dose and plasma concentration, regardless of whether PER was used as monotherapy or add-on therapy. The retention rates were 77.7% and 65. 1% at 12 and 24 months, respectively. Drug-related adverse events occurred in 45.0% of the patients and were mostly manageable.ConclusionPER effectively reduced seizure frequency in Chinese patients with FOS, particularly in those with SRE, over a 24-month period. The treatment was well-tolerated and had a clear linear dose-plasma concentration relationship

The clinical features of familial focal epilepsy with variable foci and NPRL3 gene variant

Objective Familial focal epilepsy with variable foci (FFEVF) is a rare type of focal epilepsy syndrome; it is associated with NPRL3 variant. However, relevant reports are rare in China. We aimed to analyze the clinical features of Chinese patients with FFEVF to understand further the differences between various NPRL3 variants and explored the effect of NPRL3 variant on mRNA. Methods We ran a full workup on a family with FFEVF (four patients, one healthy member): an inquiry of medical history, cranial magnetic resonance imaging (MRI), electroencephalogram (EEG), and whole exon sequencing. Their clinical features were compared with those of other FFEVF patients in published reports. The mRNA splicing changes were analyzed quantitatively and qualitatively using real-time quantitative—polymerase chain reaction (q-PCR) and reverse transcription (RT)-PCR and compared between our patients and healthy individuals. Results Patients with NPRL3: c.1137dupT variant had a wide range of onset age (4 months to 31 years), diverse seizure types, variable foci (frontal lobe/temporal lobe), different seizure times (day/night) and frequencies (monthly/seldom/every day), different therapeutic effects (refractory epilepsy/almost seizure free), normal MRI, and abnormal EEG (epileptiform discharge, slow wave). The phenotypic spectrum with different NPRL3 variants was either similar or different. Significantly different relative quantities of mRNA were found between patients and healthy individuals in real-time qPCR. Abnormal splicing was observed in patients compared with healthy individual in RT-PCR. Despite having the same gene variant, different family members had different mRNA splicing, possibly causing different phenotypes. Conclusion The clinical features of FFEVF varied, and auxiliary inspection was atypical. NPRL3: c.1137dupT could change the relative quantity of mRNA and cause abnormal splicing, which might produce different phenotypes in different family members

The clinical features of familial focal epilepsy with variable foci and NPRL3 gene variant.

ObjectiveFamilial focal epilepsy with variable foci (FFEVF) is a rare type of focal epilepsy syndrome; it is associated with NPRL3 variant. However, relevant reports are rare in China. We aimed to analyze the clinical features of Chinese patients with FFEVF to understand further the differences between various NPRL3 variants and explored the effect of NPRL3 variant on mRNA.MethodsWe ran a full workup on a family with FFEVF (four patients, one healthy member): an inquiry of medical history, cranial magnetic resonance imaging (MRI), electroencephalogram (EEG), and whole exon sequencing. Their clinical features were compared with those of other FFEVF patients in published reports. The mRNA splicing changes were analyzed quantitatively and qualitatively using real-time quantitative-polymerase chain reaction (q-PCR) and reverse transcription (RT)-PCR and compared between our patients and healthy individuals.ResultsPatients with NPRL3: c.1137dupT variant had a wide range of onset age (4 months to 31 years), diverse seizure types, variable foci (frontal lobe/temporal lobe), different seizure times (day/night) and frequencies (monthly/seldom/every day), different therapeutic effects (refractory epilepsy/almost seizure free), normal MRI, and abnormal EEG (epileptiform discharge, slow wave). The phenotypic spectrum with different NPRL3 variants was either similar or different. Significantly different relative quantities of mRNA were found between patients and healthy individuals in real-time qPCR. Abnormal splicing was observed in patients compared with healthy individual in RT-PCR. Despite having the same gene variant, different family members had different mRNA splicing, possibly causing different phenotypes.ConclusionThe clinical features of FFEVF varied, and auxiliary inspection was atypical. NPRL3: c.1137dupT could change the relative quantity of mRNA and cause abnormal splicing, which might produce different phenotypes in different family members

Relative expression level of <i>NPRl3</i>.

Compared with the CK Group, the expression levels of I1, III2, and II5 in the study group were 21, 27, and 9 times, respectively.</p

Pedigree.

The family included several members presenting with focal epilepsy. Affected individuals are shaded and asymptomatic individuals are in white. Arrow indicates the proband.</p