Microfluidic Quantitative Polymerase Chain Reaction Research Articles

Microbial assessment of water, sanitation, and hygiene (WaSH) in temporary and permanent settlements two years after Nepal 2015 earthquake

Disaster-induced displacement often causes people to live in temporary settlements that have limited infrastructure and access to water, sanitation, and hygiene (WaSH). Reducing the risk of diarrheal diseases in such situations requires knowing how housing influences the presence of pathogens in water and the interaction between human settlements and exposure to pathogens. A cross-sectional study was conducted in May 2017 in two communities hard-hit by the Nepal 2015 earthquake: one recovered with newly reconstructed houses, and one recovered with residents still living in sheet metal temporary shelters constructed after the earthquake. We collected 60 water (30 drinking water and 30 cleaning water), 30 hand rinse, and 90 environmental swab samples (30 toilet handles, 30 utensils, and 30 water vessels) from selected households in each location and quantified 22 bacterial pathogens using microfluidic quantitative polymerase chain reaction (mfqPCR). A total of 59 samples were randomly selected for amplicon-based sequencing of the 16S rRNA, and it identified bacterial community profiles between these two settlements and their association with target genes of pathogenic bacteria. Target genes like uidA of Escherichia coli and the mip gene of Legionella pnuemophila showed significantly high frequency in specific sample types in temporary settlements than in permanent settlements. A significantly high concentration was observed in temporary settlements for Enterococcus spp. and S. typhimurium, specifically in swab samples. There was a sharp distinction of microbial community profiles between water and hand rinse samples with environmental swab samples, with a large abundance of potentially pathogenic bacteria in swab samples in both settlements. This observation highlighted that fomite could be an important transmission route for pathogens in rural settings and designing key interventions to target different stages of transmission pathways is essential. Overall findings from this study suggest that the recovered settlement with higher quality housing may be less impacted by fecal contamination than recovering settlements and that interventions should be designed to disrupt multiple transmission pathways to reduce pathogen exposure.

Differences in Inflammatory Pathways Between Dutch South Asians vs Dutch Europids With Type 2 Diabetes.

South Asian individuals are more prone to develop type 2 diabetes (T2D) coinciding with earlier complications than Europids. While inflammation plays a central role in the development and progression of T2D, this factor is still underexplored in South Asians. This work aimed to study whether circulating messenger RNA (mRNA) transcripts of immune genes are different between South Asian compared with Europid patients with T2D. A secondary analysis was conducted of 2 randomized controlled trials of Dutch South Asian (n = 45; age: 55 ± 10 years, body mass index [BMI]: 29 ± 4 kg/m2) and Dutch Europid (n = 44; age: 60 ± 7 years, BMI: 32 ± 4 kg/m2) patients with T2D. Main outcome measures included mRNA transcripts of 182 immune genes (microfluidic quantitative polymerase chain reaction; Fluidigm Inc) in fasted whole-blood, ingenuity pathway analyses (Qiagen). South Asians, compared to Europids, had higher mRNA levels of B-cell markers (CD19, CD79A, CD79B, CR2, CXCR5, IGHD, MS4A1, PAX5; all fold change > 1.3, false discovery rate [FDR] < 0.008) and interferon (IFN)-signaling genes (CD274, GBP1, GBP2, GBP5, FCGR1A/B/CP, IFI16, IFIT3, IFITM1, IFITM3, TAP1; all FC > 1.2, FDR < 0.05). In South Asians, the IFN signaling pathway was the top canonical pathway (z score 2.6; P < .001) and this was accompanied by higher plasma IFN-γ levels (FC = 1.5, FDR = 0.01). Notably, the ethnic difference in gene expression was larger for women (20/182 [11%]) than men (2/182 [1%]). South Asian patients with T2D show a more activated IFN-signaling pathway compared to Europid patients with T2D, which is more pronounced in women than men. We speculate that a more activated IFN-signaling pathway may contribute to the more rapid progression of T2D in South Asian compared with Europid individuals.

Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests.

One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets in this study. A sequence region including primers and probes for quantitative PCR was designed, and a 10 bp sequence was inserted to allow the RM to be distinguished from the original transgene sequences. The sequences of individual detection sites were then connected for 12 genes and cloned into a single plasmid vector. We performed fragment size analysis to distinguish between the PCR products of the original transgene sequence and those of the RM, enabling identification of RM contamination. PTCs diluted to 10,000, 1,000, 100, and 10 copies/µl with horse genomic DNA from RM were stably stored at 4°C for 1 year. As digital PCR enabled absolute quantification, the designed substances can serve as an RM. These findings indicate that the RM design and storage conditions were suitable for gene doping tests using MFQPCR.

Downregulated Gene Expression Spectrum and Immune Responses Changed During the Disease Progression in Patients With COVID-19.

BackgroundThe World Health Organization characterizes novel coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a pandemic. Here, we investigated the clinical, cytokine levels; T-cell proportion; and related gene expression occurring in patients with COVID-19 on admission and after initial treatment.MethodsEleven patients diagnosed with COVID-19 with similar initial treatment regimens were enrolled in the hospital. Plasma cytokine, peripheral T cell proportions, and microfluidic quantitative polymerase chain reaction analyses for gene expression were conducted.ResultsFive patients with mild and 6 with severe disease were included. Cough and fever were the primary symptoms in the 11 COVID-19 cases. Older age, higher neutrophil count, and higher C-reactive protein levels were found in severe cases. IL-10 level significantly varied with disease progression and treatment. Decreased T-cell proportions were observed in patients with COVID-19, especially in severe cases, and all were returned to normal in patients with mild disease after initial treatment, but only CD4+ T cells returned to normal in severe cases. The number of differentially expressed genes (DEGs) increased with the disease progression, and decreased after initial treatment. All downregulated DEGs in severe cases mainly involved Th17-cell differentiation, cytokine-mediated signaling pathways, and T-cell activation. After initial treatment in severe cases, MAP2K7 and SOS1 were upregulated relative to that on admission.ConclusionsOur findings show that a decreased T-cell proportion with downregulated gene expression related to T-cell activation and differentiation occurred in patients with severe COVID-19, which may help to provide effective treatment strategies for COVID-19.

Antibiotic Resistance Genes in Freshwater Trout Farms in a Watershed in Chile.

Point sources such as wastewater treatment plants, terrestrial agriculture, and aquaculture may release antibiotic residues, antibiotic resistant bacteria, and antibiotic resistance genes (ARGs) into aquatic ecosystems. However, there is a lack of quantitative studies attributing environmental ARG abundance to specific sources. The goal of this study was to evaluate the role of freshwater trout farms in the release and dissemination of ARGs into the environment. Sediment samples upstream and downstream from five rainbow trout farms were collected over time in southern Chile. A microfluidic quantitative polymerase chain reaction approach was used to quantify an ARG array covering different mechanisms of resistance, and data were analyzed using principal component analysis (PCA) and linear mixed regression models. Surveys were also conducted to obtain information about management practices, including antibiotic use, at the farms. Florfenicol and oxytetracycline were used at these farms, although at different rates. A total of 93 samples were analyzed. In the PCA, , , , , (A), (B), (C), (W), and grouped together. A statistically significant increase in abundance of , , , and several genes was found downstream from the farms compared with upstream sites, and retention ponds had the highest ARG abundance at each site. Antibiotic resistance gene levels returned to baseline at an average distance of 132.7 m downstream from the farms. Although results from this study indicate an influence of trout farms on the presence of ARGs in the immediate environment, the extent of their contribution to ARG dissemination is unknown and deserves further investigation.

A Microfluidic Quantitative Polymerase Chain Reaction Method for the Simultaneous Analysis of Dozens of Antibiotic Resistance and Heavy Metal Resistance Genes

This study developed, optimized, and demonstrated a microfluidic quantitative polymerase chain reaction (MF-qPCR) method for the simultaneous quantification of 39 antibiotic resistance genes (ARGs), five heavy metal resistance genes, three genes encoding the integrase of three different classes of integrons, and 16S rRNA genes (used as a measure of total bacterial biomass). Because the volume of the template is much smaller with MF-qPCR (a few nanoliters) than with conventional qPCR, a preamplification step was needed to improve the sensitivity and the limits of quantification of the MF-qPCR method to be similar to those of conventional qPCR. The MF-qPCR method was successfully demonstrated on untreated municipal wastewater, treated municipal wastewater, and drinking water samples. The treated municipal wastewater samples had higher concentrations of all genes compared to those in the drinking water samples. Similarly, the untreated municipal wastewater samples had higher concentrations for all but one of...

Quantification of multiple waterborne pathogens in drinking water, drainage channels, and surface water in Kampala, Uganda, during seasonal variation.

Longitudinal water quality monitoring is important for understanding seasonal variations in water quality, waterborne disease transmission, and future implications for climate change and public health. In this study, microfluidic quantitative polymerase chain reaction (MFQPCR) was used to quantify genes from pathogens commonly associated with human intestinal infections in water collected from protected springs, a public tap, drainage channels, and surface water in Kampala, Uganda, from November 2014 to May 2015. The differences in relative abundance of genes during the wet and dry seasons were also assessed. All water sources tested contained multiple genes from pathogenic microorganisms, with drainage channels and surface waters containing a higher abundance of genes as compared to protected spring and the public tap water. Genes detected represented the presence of enterohemorrhagic Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, and enterovirus. There was an increased presence of pathogenic genes in drainage channels during the wet season when compared to the dry season. In contrast, surface water and drinking water sources contained little seasonal variation in the quantity of microbes assayed. These results suggest that individual water source types respond uniquely to seasonal variability and that human interaction with contaminated drainage waters, rather than direct ingestion of contaminated water, may be a more important contributor to waterborne disease transmission. Furthermore, future work in monitoring seasonal variations in water quality should focus on understanding the baseline influences of any one particular water source given their unique complexities.

Application of a Microfluidic Quantitative Polymerase Chain Reaction Technique To Monitor Bacterial Pathogens in Beach Water and Complex Environmental Matrices

Microfluidic quantitative polymerase chain reaction (MFQPCR) and conventional quantitative polymerase chain reaction methods were compared side by side in detecting and quantifying 19 genetic markers associated with Escherichia coli and select bacterial pathogens in algae, beach sand, and water from Lake Michigan. Enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli, Salmonella spp., Campylobacter jejuni, and Clostridium perfringens were among the pathogens tested. Of the pathogenic markers, eaeA that encodes intimin in EPEC was detected in all sample types: water (5%), detached/floating algae (42%), exposed/stranded algae (43%), sand below exposed algae (27%), and nearshore sand with no algae (22%). Other pathogenic markers, however, were detected sporadically. Despite comparable results from the two methods for the genetic markers tested in this study, the MFQPCR method may be superior, with the advantage of detecting and quantifying multiple pathogens simultaneously in environmental matrices.

Transcriptional Perturbations in Graft Rejection.

Understanding the regulatory interplay of relevant microRNAs (miRNAs) and messenger RNAs (mRNAs) in the rejecting allograft will result in a better understanding of the molecular pathophysiology of alloimmune injury. One hundred sixty-seven allograft biopsies, with (n = 47) and without (n = 120) central histology for Banff scored acute rejection (AR), were transcriptionally profiled for mRNA and miRNA by whole genome microarrays and multiplexed microfluidic quantitative polymerase chain reaction, respectively. A customized database was curated (GO-Elite) and used to identify AR-specific dysregulated mRNAs and the role of perturbations of their relevant miRNAs targets during AR. The AR-specific changes in 1035 specific mRNAs were mirrored by AR-specific perturbations in 9 relevant miRNAs as predicted by Go-Elite and were regulated specifically by p53 and forkhead box P3. Infiltrating lymphocytes and the renal tubules drove the miRNA tissue pertubations in rejection, involving message degradation and transcriptional/translational activation. The expression of many of these miRNAs significantly associated with the intensity of the Banff-scored interstitial inflammation and tubulitis. There is a highly regulated interplay between specific mRNA/miRNAs in allograft rejection that drive both immune-mediated injury and tissue repair during AR.