Same-Day Bilateral Intravitreal Dexamethasone Implants for the Treatment of Diabetic Macular Edema.

INTRODUCTION: The aim of this study was to evaluate the outcomes and complications associated with the use of same-day bilateral intravitreal dexamethasone (DEX) implants for the treatment of diabetic macular edema (DME). METHODS: This retrospective analysis of an open-label, multicenter, consecutive case series included 130 eyes of 65 patients with bilateral DME who were treated with intravitreal DEX implants. The patients were divided into two groups: a control group (comprising 40 eyes treated with an alternating unilateral regimen) and a study group (comprising 90 eyes treated with concomitant bilateral DEX implants). All patients were followed up monthly after implantation. The changes in best-corrected visual acuity (BCVA) and central retinal thickness (CRT) from baseline to sixth month after implantation, and ocular adverse effects such as intraocular pressure, cataract, and tolerability of bilateral implantation were reviewed. The primary endpoint was to assess the safety of the same-day bilateral treatment protocol. The secondary endpoints focused on evaluating the functional and anatomical changes associated with bilateral simultaneous or alternating implantations. RESULTS: At 6 months after implantation, mean BCVA increased and CRT decreased in both groups. Moreover, no serious ocular adverse effects were observed. In addition, no differences were observed between the two groups in the number of patients who required extra follow-up visits or the number of extra visits made in addition to the treatment schedule. CONCLUSIONS: Same-day bilateral intravitreal DEX implants are associated with a low complication rate and are well tolerated by patients. This safe practice may optimize efficiency and reduce the burden on both the health-care system and patients, when used to treat bilateral DME.

The Post-Translational Modification Networking in WNK-Centric Hypertension Regulation and Electrolyte Homeostasis.

The with-no-lysine (WNK) kinase family, comprising four serine-threonine protein kinases (WNK1-4), were first linked to hypertension due to their mutations in association with pseudohypoaldosteronism type II (PHAII). WNK kinases regulate crucial blood pressure regulators, SPAK/OSR1, to mediate the post-translational modifications (PTMs) of their downstream ion channel substrates, such as sodium chloride co-transporter (NCC), epithelial sodium chloride (ENaC), renal outer medullary potassium channel (ROMK), and Na/K/2Cl co-transporters (NKCCs). In this review, we summarize the molecular pathways dysregulating the WNKs and their downstream target renal ion transporters. We summarize each of the genetic variants of WNK kinases and the small molecule inhibitors that have been discovered to regulate blood pressure via WNK-triggered PTM cascades.

Widen the Applicability of a Convolutional Neural-Network-Assisted Glaucoma Detection Algorithm of Limited Training Images across Different Datasets.

Automated glaucoma detection using deep learning may increase the diagnostic rate of glaucoma to prevent blindness, but generalizable models are currently unavailable despite the use of huge training datasets. This study aims to evaluate the performance of a convolutional neural network (CNN) classifier trained with a limited number of high-quality fundus images in detecting glaucoma and methods to improve its performance across different datasets. A CNN classifier was constructed using EfficientNet B3 and 944 images collected from one medical center (core model) and externally validated using three datasets. The performance of the core model was compared with (1) the integrated model constructed by using all training images from the four datasets and (2) the dataset-specific model built by fine-tuning the core model with training images from the external datasets. The diagnostic accuracy of the core model was 95.62% but dropped to ranges of 52.5-80.0% on the external datasets. Dataset-specific models exhibited superior diagnostic performance on the external datasets compared to other models, with a diagnostic accuracy of 87.50-92.5%. The findings suggest that dataset-specific tuning of the core CNN classifier effectively improves its applicability across different datasets when increasing training images fails to achieve generalization.

Therapeutic effect of simultaneous intravitreal dexamethasone and aflibercept on diabetic macular edema.

AIMS: To report the effect of simultaneous intravitreal dexamethasone (DEX) and aflibercept for the treatment of diabetic macular edema (DME). METHODS: This retrospective analysis of an open-label, multicenter, consecutive case series included 102 eyes of 81 patients with DME. Patients were selected into two groups. The control group consisted of 50 eyes treated with aflibercept alone, and the combination group consisted of 52 eyes treated with simultaneous DEX implant and aflibercept injection. The primary endpoints were changes in best-corrected visual acuity (BCVA) and central retinal thickness (CRT) from baseline to month 6. The secondary endpoint was the interval of retreatment. RESULTS: Baseline BCVA increased and CRT decreased at 6 months in both groups. Pseudophakic eyes in the combination group exhibited significantly greater BCVA improvement compared with phakic eyes (p = 0.031). Fewer intravitreal treatments were required for eyes treated with combination therapy than for those treated with aflibercept alone (1.56 ± 0.54 vs. 4.04 ± 1.26, p < .0001), with a mean retreatment interval of 3.66 ± 0.69 months. CONCLUSIONS: Simultaneous intravitreal DEX and aflibercept achieved non-inferior improvement of visual and anatomic outcomes compared with aflibercept alone for DME, but exhibited a significantly longer treatment interval and superior visual outcome in pseudophakic eyes. This therapeutic approach is considered a valid strategy for treating DME in the era of COVID-19.

Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample.

Acanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

Prevalence, enterotoxin-gene profiles, antimicrobial resistance, and genetic diversity of Bacillus cereus group in aquatic environments and shellfish.

Bacterial strains of the Bacillus cereus group produce various toxins that cause diarrheal and emetic food poisoning. In this study, five main oyster farming areas and 15 fishing ports in Taiwan were examined for the status of B. cereus group bacteria inhabiting seawater and shellfish. On average, bacteria of the B. cereus group were detected in 32.6% of the seawater samples (n = 89) and 2.5% of the oysters (n = 81) in the oyster farming areas and in 7.9% of the seawater samples (n = 202) and 0.68% of the shellfish products (n = 292) in fishing ports. To trace the potential source of B. cereus group bacteria in intertidal oyster farming areas, we simultaneously explored their terrestrial river basins. In total, 44 B. cereus group strains were purified and cultured from water and shellfish for the analysis of virulence genes, panC gene typing, antibiotic susceptibility testing, and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) fingerprinting. The enterotoxin-coding genes nheABC, hblCDA, entFM, cytK-1, and cytK-2 were detected in 70.4%, 84.1%, 97.7%, 72.7%, and 75% of the total isolates, among which 40.9% carried all these genes. According to panC gene analysis, the dominant isolates belonged to the panC group IV. In antibiotic susceptibility tests, most B. cereus group isolates were resistant to ampicillin (97.7%) and sulfamethoxazole/trimethoprim (100%). The percentage of multidrug resistant B. cereus group isolates was 34.6%. Finally, the 44 B. cereus group isolates were classified into 43 types and categorized into five clusters using ERIC-PCR fingerprinting. The B. cereus group isolates from different oyster farming areas were concentrated within the two main clusters; however, those from river basins displayed a wide genetic diversity, indicating the presence of multiple sources of B. cereus group bacteria in river basins.

Swimming Pool-Associated Vittaforma-Like Microsporidia Linked to Microsporidial Keratoconjunctivitis Outbreak, Taiwan.

We analyzed 2 batches of environmental samples after a microsporidial keratoconjunctivitis outbreak in Taiwan. Results indicated a transmission route from a parking lot to a foot washing pool to a swimming pool and suggested that accumulation of mud in the foot washing pool during the rainy season might be a risk factor.

Characterization and functional correlation of multiple imaging modalities with focal choroidal excavation.

BACKGROUND: To investigate the clinical manifestations and imaging features of near-infrared autofluorescence (NIA), infrared reflectance (IR), fundus autofluorescence (FAF), indocyanine green angiography (ICGA) and fluorescein angiography (FAG) in the detection of patients with focal choroidal excavation (FCE) identified by cross-sectional spectral-domain optical coherence tomography (SD-OCT). METHODS: This retrospective cross-sectional study included 12 eyes of 10 Taiwanese patients with FCE diagnosed by SD-OCT. The areas and depths of FCE in serial cross-sectional and en-face OCT were compared in different imaging modalities. NIA, IR, FAF, ICGA and FAG images were obtained. Best corrected visual acuity, subjective distortion area in the Amsler grid and history of maculopathies were also recorded. RESULTS: In areas where the choroid started to excavate as shown in SD-OCT, hypo-autofluorescence in NIA was noted. The area of hypo-fluorescence in NIA of all the FCE lesions showed good correlation with the size. The area of FCE was associated with complications such as choroidal neovascularization and central serous chorioretinopathy (p = 0.014, d.f = 1) and the volume (NIA area × Depth measured by SD-OCT × 1/3) was associated with subjective distortion strongly (p = 0.051, Spearman's correlation = 0.600). CONCLUSION: Among all image modalities, NIA was the most sensitive tool in area measurement of FCE and peripheral lesion detection. Also, the volume of FCE was associated with subjective distortion and the area was related to complications. Recording the area and volume of FCE could play an important role in monitoring complications.

Application of modified JDP-DGGE-based molecular genotyping method to predict Acanthamoeba genotype and to analyse community diversity in aquatic environments.

Acanthamoeba spp. are ubiquitous, opportunistic potential human pathogens, causing granulomatous amoebic encephalitis and keratitis. They are classified as protozoa, and they include at least 20 different genotypes (T1-T20) based on variation in the 18S rRNA gene. Acanthamoeba spp. are diverse in their production of toxins and in their ability to resist environmental factors. Therefore, it is necessary to develop a rapid genotyping method for Acanthamoeba spp. in aquatic environments. Although the denaturing gradient gel electrophoresis (DGGE) method for analysing microbial genotypes is potentially useful for rapid identification of aquatic environmental species, the technique has been compromised by artificial DGGE profiles in which many DNA fragments of identical sequences are segregated and displayed as different bands. The results indicate that PCR-DGGE genotyping with a GC clamp results in many segregated weaker bands of identical DNA sequences. In contrast, PCR-DGGE genotyping without a GC clamp displays genotype-dependent patterns in the major bands. Thus, DGGE without a GC clamp was performed to compare genotyping efficiency for Acanthamoeba in 21 water samples from rivers and reservoirs in Taiwan. Among them, four samples were found to demonstrate a banding pattern with more than one major band, and these band profiles of major bands were identical to those of positive controls. DNA cloning further confirmed that the sequences of the major bands were identical. In conclusion, more than two genotypes of Acanthamoeba in the four samples were identified by this method, suggesting that PCR-DGGE genotyping without a GC clamp is a useful approach for studying the diversity of Acanthamoeba communities. Graphical abstract.

First detection of enteric adenoviruses genotype 41 in recreation spring areas of Taiwan.

Human adenoviruses (HAdVs) are DNA viruses found in recreational water, such as water parks and swimming pools. Human adenovirus 41 (HAdV-41) is the most common serotype detected and is a leading cause of acute diarrheal disease. The focus of this study is to determine the prevalence of HAdVs in hot springs. Of 57 samples collected from four different geological sites, 16 samples have shown evidence of HAdVs (28.1%). HAdV-41 and porcine adenovirus 5 (PAdV-5) were the two types isolated, with a greater frequency of HAdV-41, which in other settings has been associated with acute diarrhea. The highest occurrence was found in private hot tubs/Yuya (37.5%), followed by an outlet of hot springs (30.8%); public pools and foot pools shared the same detection rate of 21.4% (3/14). However, there was no evidence supporting a link between water quality indicators and HAdV detection rate. From a phylogenic analysis and BLAST against the NCBI database, it was concluded that HAdV-41 obtained from hot spring areas are closely related to global environmental genotypes.

Surveillance of Vittaforma corneae in hot springs by a small-volume procedure.

Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence supporting the need for proper control and regulations at hot spring recreational waters in order to avoid health risks from this pathogen. Finally, we have developed a small volume procedure for detecting V. corneae in water samples and this has proved to be very useful.

Nested-PCR and TaqMan real-time quantitative PCR assays for human adenoviruses in environmental waters.

Human adenovirus (HAdV) infections can occur throughout the year. Cases of HAdV-associated respiratory disease have been more common in the late winter, spring, and early summer. In this study, to provide viral pollution data for further epidemiological studies and governmental actions, the presence of HAdV in the aquatic environment was quantitatively surveyed in the summer. This study was conducted to compare the efficiencies of nested-PCR (polymerase chain reaction) and qPCR (quantitative PCR) for detecting HAdV in environmental waters. A total of 73 water samples were collected from Puzi River in Taiwan and subjected to virus concentration methods. In the results, qPCR had much better efficiency for specifying the pathogen in river sample. HAdV41 was detected most frequently in the river water sample (10.9%). The estimated HAdV concentrations ranged between 6.75 × 10(2) and 2.04 × 10(9) genome copies/L. Significant difference was also found in heterotrophic plate counts, conductivity, water temperature, and water turbidity between presence/absence of HAdV. HAdV in the Puzi River may pose a significant health risk.

Seasonal difference of human adenoviruses in a subtropical river basin based on 1-year monthly survey.

In this study, the seasonal difference and the observable presence/absence of human adenovirus (HAdV) in the Puzih River basin in Taiwan was investigated. A total of 288 water samples were collected from 24 sites from March 2014 to February 2015. Human AdV analysis of sample was subjected to viral concentration using a GN-6 Metricel® filter, followed by DNA extraction, nested-PCR, and qPCR. Human AdV was detected in 34.3 % (99/288) of the entire river water sample. A higher percentage of HAdV (76.4 %) was obtained during the winter. The HAdV median concentration was relatively high in fall (1.4 × 10(3) copies/L) and winter (2.8 × 10(3) copies/L). Significant difference and correlation were found between the seasonal variation of HAdV and water quality parameters, including heterotrophic plate count, total coliform, water temperature, and turbidity. The most frequently identified HAdV (subgenus F) serotype was 41. Human AdV-41 is the main cause of gastroenteritis and should be considered for associated human health risk potential in the Puzih River basin.

Surveillance of parasitic Legionella in surface waters by using immunomagnetic separation and amoebae enrichment.

Free-living amoebae (FLA) are potential reservoirs of Legionella in aquatic environments. However, the parasitic relationship between various Legionella and amoebae remains unclear. In this study, surface water samples were gathered from two rivers for evaluating parasitic Legionella. Warmer water temperature is critical to the existence of Legionella. This result suggests that amoebae may be helpful in maintaining Legionella in natural environments because warmer temperatures could enhance parasitisation of Legionella in amoebae. We next used immunomagnetic separation (IMS) to identify extracellular Legionella and remove most free Legionella before detecting the parasitic ones in selectively enriched amoebae. Legionella pneumophila was detected in all the approaches, confirming that the pathogen is a facultative amoebae parasite. By contrast, two obligate amoebae parasites, Legionella-like amoebal pathogens (LLAPs) 8 and 9, were detected only in enriched amoebae. However, several uncultured Legionella were detected only in the extracellular samples. Because the presence of potential hosts, namely Vermamoeba vermiformis, Acanthamoeba spp. and Naegleria gruberi, was confirmed in the samples that contained intracellular Legionella, uncultured Legionella may survive independently of amoebae. Immunomagnetic separation and amoebae enrichment may have referential value for detecting parasitic Legionella in surface waters.

Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

Antibiotic resistance pattern and gene expression of non-typhoid Salmonella in riversheds.

In this study, antibiotic resistance and major phenol and genotypes of non-typhoid Salmonella spp. from riversheds in Taiwan were examined. In 236 water samples tested, 54 (22.9%) contained Salmonella spp. Fifteen Salmonella serovars were identified from the Salmonella isolates, and some common serovars are associated with infections of human and livestock, including Albany (27.8%), Newport (14.8%), Bareilly (13.0%), Derby (11.1%), and Typhimurium (7.4%). Various environmental factors may also affect the presence and proportion of different serovars in the receiving waters. In contrast, serovars with narrower range of hosts, e.g., Dublin, were rarely detected. The Salmonella isolates were subjected to eight antibiotics for drug resistance, and 51.9% of the samples were resistant to at least one tested antibiotics. Tetracycline and sulfadiazine were the two most ineffective antibiotics against the Salmonella isolates, and the results were indicative of long-term antibiotics abuse as fodder supplements in animal husbandry. The more commonly detected serovars such as Albany, Derby, and Typhimurium were also more likely to be resistant to multiple antibiotics. Finally, a significant correlation was observed between resistance to chloramphenicol and the resistance gene cmlA, suggesting that the resistance genotypes could persist in the environment even long after prohibition of the drug use. The high prevalence of antibiotic-resistant Salmonella spp. infers elevated infection risks that must be further examined.

Seasonal distribution of potentially pathogenic Acanthamoeba species from drinking water reservoirs in Taiwan.

In order to detect the presence/absence of Acanthamoeba along with geographical variations, water quality variations and seasonal change of Acanthamoeba in Taiwan was investigated by 18S ribosomal RNA (rRNA) gene TaqMan quantitative real-time PCR. Samples were collected quarterly at 19 drinking water reservoir sites from November 2012 to August 2013. Acanthamoeba was detected in 39.5 % (30/76) of the water sample, and the detection rate was 63.2 % (12/19) from samples collected in autumn. The average concentration of Acanthamoeba was 3.59 × 10(4) copies/L. For geographic distribution, the detection rate for Acanthamoeba at the northern region was higher than the central and southern regions in all seasons. Results of Spearman rank test revealed that heterotrophic plate count (HPC) had a negative correlation (R = -0.502), while dissolved oxygen (DO) had a positive correlation (R = 0.463) in summer. Significant differences were found only between the presence/absence of Acanthamoeba and HPC in summer (Mann-Whitney U test, P < 0.05). T2 and T4 genotypes of Acanthamoeba were identified, and T4 was the most commonly identified Acanthamoeba genotypes. The presence of Acanthamoeba in reservoirs presented a potential public health threat and should be further examined.

Seasonal variation of Legionella in Taiwan's reservoir and its relationships with environmental factors.

In this study, the presence of Legionella in major water reservoirs of Taiwan was examined with respect to seasonal variation, geographical variation, and water quality parameters using TaqMan real-time qPCR. Water samples were collected quarterly at 19 reservoirs in Taiwan between November 2012 and August 2013. The detection rate for Legionella was 35.5% (27/76), and Legionella was detected in all seasons. The Legionella concentration was relatively high in spring and summer, reaching 3.86 × 10(8) and 7.35 × 10(8) cells/L, respectively. By sampling the area, Legionella was detected at a higher proportion in reservoirs in the northern and southern areas, and the difference was consistent in all seasons. Significant association was found between detection of Legionella and various water quality parameters, including conductivity, chlorophyll a, and dissolved oxygen (Mann-Whitney U test, P < 0.05). Results of Spearman rank test showed negative correlation for Legionella detection with pH (P = 0.030, R = -0.497) and dissolved oxygen (P = 0.007, R = -0.596) in fall and positive correlation with Carlson's trophic state index (P = 0.049, R = 0.457) in spring. The identified species included Legionella pneumophila and Legionella drancourtii. The detection of Legionella in reservoirs was indicative of a potential public health risk and should be further evaluated.

Prevalence, quantification, and typing of human adenoviruses detected in river water in Taiwan.

The prevalence of human adenoviruses (HAdV) in river waters was investigated in this study. Water samples were collected from 13 rivers in Taiwan, concentrated, and assessed for the presence of HAdVs using nested polymerase chain reaction (PCR). Human AdV positive samples were then subjected to real-time PCR (qPCR) to quantify the viral genomes and further subjected to primer-based genotyping to identify the various serotypes present. For each water sample, several water quality parameters were evaluated, including heterotrophic plate count, total coliform, Escherichia coli, water temperature, pH, conductivity, and dissolved oxygen. Among the 13 rivers examined, four rivers (30.8 %) were found to contain HAdVs. The major genotype was F species HAdV serotype 41. The mean HAdVs concentrations ranged from 6.10 × 10(2) to 8.51 × 10(2) copies/L. No significant differences were observed between the presence of HAdVs, and all of the water quality parameters evaluated (heterotrophic plate count, total coliform, E. coli, water temperature, pH, conductivity, and dissolved oxygen). Given the potential health risks posed by the presence of enteric viruses in environmental waters, further assessment is desirable with respect to possible sources, virus transport, and survival of viruses in the aquatic environment.

Evaluation of diarrheagenic E. coli in riversheds by quantitative PCR in combination with enrichment.

Diarrheagenic Escherichia coli (DEC) is a group of the most common agents of diarrhea. Highly virulent DEC strains could cause illness with dozens of organisms. Waterborne DEC may be detected using polymerase chain reaction (PCR); however, environmental contaminants can interfere with PCR reaction, thus causing the prevalence of DEC to be underestimated. In this study, we propose an approach to efficiently quantify trace amounts of DEC. An enrichment procedure was performed to amplify total E. coli including DEC in the water samples. By normalizing the number of pathotype-specific genes to the amplification rate of a housekeeping gene in all E. coli, the quantity of DEC in original samples could be assessed. This method allows detection of trace amounts of DEC in receiving waters. The results showed that the presence of DEC in water samples was partially associated with riverside settlement. The DEC concentration was substantially higher at a few sampling sites, suggesting that evaluation of DEC along the river may help identify previously unknown pollution sources. Although the sustainability of DEC in the receiving waters may be low, the risk of DEC infection from the watershed warrants further examination.