Methicillin-Resistant Staphylococcus aureus Contamination of Frequently Touched Objects in Intensive Care Units: Potential Threat of Nosocomial Infections.

Background: Bacterial contamination in intensive care units is an important risk factor associated with increasing incidences of nosocomial infections. This study was conducted to study the bacterial colonization on commonly touched objects of intensive care units and antibiotic resistance pattern of bacterial isolates. Methods: This study was conducted in different intensive care units of Manipal Teaching Hospital, Pokhara, Nepal. A total of 235 swabs were collected from surfaces of bed rails, monitors, door handles, IV stands, telephone sets, nursing stations, medicine trolleys, sphygmomanometers, wash basin taps, dressing drums, stethoscopes, pulse oximeters, ventilators, defibrillators, and stretchers. Isolation, identification, and antibiotic susceptibility tests of the bacteria were performed following standard microbiological techniques. Results: Of 235 samples, bacterial growth was observed in 152 samples. A total of 90 samples of Staphylococcus aureus were isolated from 235 samples. Most of the sampling sites included in this study were found contaminated with S. aureus. The highest number of S. aureus was cultured from the surface of bed rails. Of the total S. aureus isolates, 54.4% (49/90) were methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin resistance was detected among 8.1% MRSA isolates (4/49). Acinetobacter species were the commonest Gram-negative bacterial isolate. Conclusion: Bacterial contamination of the objects/instruments of the ICU was recorded to be high. The most common contaminating bacteria were S. aureus with a high percentage of MRSA and emergence of VRSA. Periodic microbiological surveillance, detection of contaminated sites, and effective decontamination methods would minimize the colonization by potential pathogens and their transmission.

Identifying the Transcriptome Signatures of Calcium Channel Blockers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: Calcium channel blockers (CCBs) are an important class of drugs in managing cardiovascular diseases. Patients usually rely on these medications for the remainder of their lives after diagnosis. Although the acute pharmacological actions of CCBs in the hearts are well-defined, little is known about the drug-specific effects on human cardiomyocyte transcriptomes and physiological alterations after long-term exposure. OBJECTIVE: This study aimed to simulate chronic CCB treatment and to examine both the functional and transcriptomic changes in human cardiomyocytes. METHODS AND RESULTS: We differentiated cardiomyocytes and generated engineered heart tissues from 3 human induced pluripotent stem cell lines and exposed them to 4 different CCBs-nifedipine, amlodipine, diltiazem, and verapamil-at their physiological serum concentrations for 2 weeks. Without inducing cell death and damage to myofilament structure, CCBs elicited line-specific inhibition on calcium kinetics and contractility. While all 4 CCBs exerted similar inhibition on calcium kinetics, verapamil applied the strongest inhibition on cardiomyocyte contractile function. By profiling cardiomyocyte transcriptome after CCB treatment, we identified little overlap in their transcriptome signatures. Verapamil is the only inhibitor that reduced the expression of contraction-related genes, such as MYH (myosin heavy chain) and troponin I, consistent with its depressive effects on contractile function. The reduction of these contraction-related genes may also explain the responsiveness of patients with hypertrophic cardiomyopathy to verapamil in managing left ventricular outflow tract obstruction. CONCLUSIONS: This is the first study to identify the transcriptome signatures of different CCBs in human cardiomyocytes. The distinct gene expression patterns suggest that although the 4 inhibitors act on the same target, they may have distinct effects on normal cardiac cell physiology.

Effects of selection and compiling strategy of substrates in column-type vertical-flow constructed wetlands on the treatment of synthetic landfill leachate containing bisphenol A.

A constructed wetland (CW) is a low-cost, eco-friendly, easy-to-maintain, and widely applicable technology for treating various pollutants in the waste landfill leachate. This study determined the effects of the selection and compiling strategy of substrates used in CWs on the treatment performance of a synthetic leachate containing bisphenol A (BPA) as a representative recalcitrant pollutant. We operated five types of lab-scale vertical-flow CWs using only gravel (CW1), a sandwich of gravel with activated carbon (CW2) or brick crumbs (CW3), and two-stage hybrid CWs using gravel in one column and activated carbon (CW4) or brick crumbs (CW5) in another to treat synthetic leachate containing BPA in a 7-d sequential batch mode for 5 weeks. CWs using activated carbon (CW2 and CW4) effectively removed ammonium nitrogen (NH4-N) (99-100%), chemical oxygen demand (COD) (93-100%), and BPA (100%), indicating that the high adsorption capacity of activated carbon was the main mechanism involved in their removal. CW5 also exhibited higher pollutant removal efficiencies (NH4-N: 94-99%, COD: 89-98%, BPA: 89-100%) than single-column CWs (CW1 and CW3) (NH4-N: 76-100%, COD: 84-100%, BPA: 51-100%). This indicates the importance of the compiling strategy along with the selection of an appropriate substrate to improve the pollutant removal capability of CWs.

A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin-binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). METHODS: Isogenic iPSC lines were generated from HCM patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing. Comprehensive phenotypic and transcriptome analyses were performed in the iPSC-CMs. RESULTS: We observed aberrant calcium handling properties with prolonged decay kinetics and elevated diastolic calcium levels in the absence of structural abnormalities or contracile dysfunction in HCM iPSC-CMs as compared to isogenic controls. The mRNA expression levels of MYBPC3 were significantly reduced in mutant iPSC-CMs, but the protein levels were comparable among isogenic iPSC-CMs, suggesting that haploinsufficiency of MYBPC3 does not contribute to the pathogenesis of HCM in vitro. Furthermore, truncated MYBPC3 peptides were not detected. At the molecular level, the nonsense-mediated decay pathway was activated, and a set of genes involved in major cardiac signaling pathways was dysregulated in HCM iPSC-CMs, indicating an HCM gene signature in vitro. Specific inhibition of the nonsense-mediated decay pathway in mutant iPSC-CMs resulted in reversal of the molecular phenotype and normalization of calcium-handling abnormalities. CONCLUSIONS: iPSC-CMs carrying MYBPC3 PTC mutations displayed aberrant calcium signaling and molecular dysregulations in the absence of significant haploinsufficiency of MYBPC3 protein. Here we provided the first evidence of the direct connection between the chronically activated nonsense-mediated decay pathway and HCM disease development.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as Models for Cardiac Channelopathies: A Primer for Non-Electrophysiologists.

Life threatening ventricular arrhythmias leading to sudden cardiac death are a major cause of morbidity and mortality. In the absence of structural heart disease, these arrhythmias, especially in the younger population, are often an outcome of genetic defects in specialized membrane proteins called ion channels. In the heart, exceptionally well-orchestrated activity of a diversity of ion channels mediates the cardiac action potential. Alterations in either the function or expression of these channels can disrupt the configuration of the action potential, leading to abnormal electrical activity of the heart that can sometimes initiate an arrhythmia. Understanding the pathophysiology of inherited arrhythmias can be challenging because of the complexity of the disorder and lack of appropriate cellular and in vivo models. Recent advances in human induced pluripotent stem cell technology have provided remarkable progress in comprehending the underlying mechanisms of ion channel disorders or channelopathies by modeling these complex arrhythmia syndromes in vitro in a dish. To fully realize the potential of induced pluripotent stem cells in elucidating the mechanistic basis and complex pathophysiology of channelopathies, it is crucial to have a basic knowledge of cardiac myocyte electrophysiology. In this review, we will discuss the role of the various ion channels in cardiac electrophysiology and the molecular and cellular mechanisms of arrhythmias, highlighting the promise of human induced pluripotent stem cell-cardiomyocytes as a model for investigating inherited arrhythmia syndromes and testing antiarrhythmic strategies. Overall, this review aims to provide a basic understanding of the electrical activity of the heart and related channelopathies, especially to clinicians or research scientists in the cardiovascular field with limited electrophysiology background.

Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells.

BACKGROUND: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. METHODS: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS MYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUS MYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUS MYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). RESULTS: The heterozygous VUS MYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS MYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. CONCLUSIONS: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.

A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases.

RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in Nepal.

This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.

Nasal and Pharyngeal Colonization by Bacterial Pathogens: A Comparative Study between Preclinical and Clinical Sciences Medical Students.

BACKGROUND: Upper respiratory tract is one of the commonest sites for microbial colonization. The colonized individuals are at risk of infections and can be a source of transmission of pathogens. Medical students are frequently exposed to a variety of infectious agents and more likely to get colonized by them. This study was aimed to determine the prevalence and to compare the colonization rates of nasal and pharyngeal bacterial pathogens among preclinical and clinical sciences medical students. METHODS: This cross-sectional study was conducted among 100 preclinical and 100 clinical sciences medical students. Isolation, identification, and antibiotic susceptibility testing of the isolates were performed by standard microbiological techniques. RESULTS: The nasal colonization by S. aureus and MRSA was 35% (70/200) and 19.5% (39/200), respectively. The nasal colonization by S. aureus and MRSA was significantly higher among clinical sciences students as compared to preclinical sciences students. Pharyngeal colonization by Haemophilus influenzae was significantly higher among clinical sciences students as compared to preclinical sciences students. The pharyngeal colonization by beta-hemolytic streptococci (nongroup A) was higher among preclinical sciences students than clinical sciences students. CONCLUSION: The nasal colonization by S. aureus and MRSA was higher among clinical sciences students. Pharyngeal colonization by potential bacterial pathogens was higher among clinical sciences students than preclinical students. Periodic screening of MRSA and potential throat pathogens of clinical sciences students and may reduce the incidences of nosocomial transmission of pathogens.

Bacterial meningitis in children under 15 years of age in Nepal.

BACKGROUND: Bacterial meningitis in children is a life-threatening problem resulting in severe morbidity and mortality. For the prompt initiation of antibacterial therapy, rapid and reliable diagnostic methods are of utmost importance. Therefore, this study was designed to find out the rate of bacterial pathogens of meningitis from suspected cases by performing conventional methods and latex agglutination. METHODS: A descriptive type of study was carried out from May 2012 to April 2013. Cerebrospinal fluid (CSF) specimens from 252 suspected cases of meningitis were subjected for Gram staining, bacterial culture and latex agglutination test. The identification of growth of bacteria was done following standard microbiological methods recommended by American Society for Microbiology. Antibiotic sensitivity testing was done by modified Kirby-Bauer disk diffusion method. RESULTS: From the total 252 suspected cases, 7.2 % bacterial meningitis was revealed by Gram staining and culture methods whereas latex agglutination method detected 5.6 %. Gram-negative organisms contributed the majority of the cases (72.2 %) with Haemophilus influenzae as the leading pathogen for meningitis. Overall, 33.3 % mortality rate was found. CONCLUSIONS: In conclusion, a significant rate of bacterial meningitis was found in this study prompting concern for national wide surveillance.

Helicobacter Pylori Infection among Patients Attending the Gastroenterology Department in Tertiary Care Hospital, Kathmandu, Nepal.

Helicobacter pylori is one of the most pathogenic organisms that cause gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma in humans. The main aim of this study was to determine the H. pylori infection among patients undergoing upper GI endoscopy and to compare the efficacy of the diagnostic method of H. pylori infection including invasive tests (biopsy-based tests like the rapid urease test (RUT), direct smear, and culture) and the noninvasive test (HpSA). A total of 100 stool samples and 200 gastric biopsy specimens were collected (2 samples from each patient) from June to November 2019. Stool samples were processed for the detection of an H. pylori stool antigen (HpSA) by a kit method. One biopsy specimen was processed for the RUT, and another was transported to the laboratory in an Eppendorf tube containing normal saline for preparation of the smear and culture. Out of 100 participants, 26% were found to be H. pylori positive by the RUT, 11% by the direct smear, 6% by the culture, and 17% by the stool antigen test. The prevalence of H. pylori infection was found to be 14%, considering at least two of the three biopsy-based tests that gave positive results. H. pylori infection was found to be higher in the age group of 46-55 years. The overall prevalence of H. pylori infection was higher in gastric ulcer cases, followed by erosive pangastritis and gastroduodenitis. Tea drinking habits and the frequency of meal consumption more than twice a day were found to be significantly associated with H. pylori infection (P < 0.05). Hence, the RUT was found to be more efficient than the direct smear and the culture method for finding H. pylori in the biopsy sample. However, none of these methods can be considered to be the gold standard alone. Thus, the RUT combined with other tests is preferable for the detection of H. pylori.

Effects of Cryopreservation on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Assessing Drug Safety Response Profiles.

Burgeoning applications of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in disease modeling, regenerative medicine, and drug screening have broadened the usage of hiPSC-CMs and entailed their long-term storage. Cryopreservation is the most common approach to store hiPSC-CMs. However, the effects of cryopreservation and recovery on hiPSC-CMs remain poorly understood. Here, we characterized the transcriptome, electro-mechanical function, and drug response of fresh hiPSC-CMs without cryopreservation and recovered hiPSC-CMs from cryopreservation. We found that recovered hiPSC-CMs showed upregulation of cell cycle genes, similar or reduced contractility, Ca2+ transients, and field potential duration. When subjected to treatment of drugs that affect electrophysiological properties, recovered hiPSC-CMs showed an altered drug response and enhanced propensity for drug-induced cardiac arrhythmic events. In conclusion, fresh and recovered hiPSC-CMs do not always show comparable molecular and physiological properties. When cryopreserved hiPSC-CMs are used for assessing drug-induced cardiac liabilities, the altered drug sensitivity needs to be considered.

Bacterial contamination of neonatal intensive care units: How safe are the neonates?

BACKGROUND: Intensive care units (ICU) are essential healthcare facility for life threatening conditions. Bacterial contamination of objects/instruments in ICU is an important source of nosocomial infections. This study is aimed to determine the level of bacterial contamination of instruments/objects which are commonly touched by healthcare workers and frequently come in contact with the neonates. METHODS: This hospital based prospective study was conducted in neonatal intensive care unit (NICU) of Manipal Teaching Hospital, Pokhara, Nepal. A total of 146 samples collected from surfaces of incubators, radiant warmers, suction tips, ventilators, stethoscopes, door handles, weighing machines, mothers' beds, phototherapy beds, laryngoscope, telephone sets, blood pressure machine, etc. formed the material of the study. Isolation, identification and antibiotic susceptibility of the bacterial isolates was performed by standard techniques. Blood culture isolates from NICU patients during the study period were compared with the environmental isolates. RESULTS: Out of 146 samples, bacterial growth was observed in 109. A total of 119 bacterial isolates were retrieved from 109 samples. Three common potential pathogens isolated were Escherichia coli (n = 27), Klebsiella species (n = 21) and Staphylococcus aureus (n = 18). Majority of E. coli and Klebsiella isolates were from incubators, suction tips and mothers' beds. Majority of S. aureus isolates were cultured from radiant warmers. Among S. aureus isolates, 33.3% (6/18) were methicillin resistant. Majority of the bacterial isolates were susceptible to gentamicin and amikacin. Common potential pathogens isolated from blood culture of NICU patients were S. aureus and Klebsiella species. CONCLUSION: High degree of bacterial contamination of objects/instruments in NICU was recorded. Isolation of potential pathogens like E. coli, Klebsiella species and S. aureus is a major threat of nosocomial infections. Blood culture data of NICU reflects possibility of nosocomial infections from contaminated sites. Gentamicin and amikacin may be used for empirical therapy in suspected cases of nosocomial infections in NICU.

Identification of 16S rRNA and Virulence-Associated Genes of Arcobacter in Water Samples in the Kathmandu Valley, Nepal.

This study aimed to determine the prevalence of Arcobacter and five associated virulence genes (cadF, ciaB, mviN, pldA, and tlyA) in water samples in the Kathmandu Valley, Nepal. A total of 286 samples were collected from deep tube wells (n = 30), rivers (n = 14), a pond (n = 1), shallow dug wells (n = 166), shallow tube wells (n = 33), springs (n = 21), and stone spouts (n = 21) in February and March (dry season) and August (wet season), 2016. Bacterial DNA was extracted from the water samples and subjected to SYBR Green-based quantitative PCR for 16S rRNA and virulence genes of Arcobacter. The 16S rRNA gene of Arcobacter was detected in 36% (40/112) of samples collected in the dry season, at concentrations ranging from 5.7 to 10.2 log copies/100 mL, and 34% (59/174) of samples collected in the wet season, at concentrations of 5.4-10.8 log copies/100 mL. No significant difference in Arcobacter 16S rRNA gene-positive results was observed between samples collected in the two seasons (p > 0.05). Seventeen (17%), 84 (84%), 19 (19%), 23 (23%), and 17 (17%) of the 99 Arcobacter 16S rRNA gene-positive samples were also positive for cadF, ciaB, mviN, pldA, and tlyA, respectively. At least one virulence gene was detected in 87 (88%) of the 99 Arcobacter 16S rRNA gene-positive samples. The presence of Arcobacter and the virulence genes in these samples illustrates the persistence of pathogenic bacteria in the environment and highlights the importance of regular monitoring of water for pathogens.

Anatomic Variations of the Right Hepatic Duct: Results and Surgical Implications From a Cadaveric Study.

BACKGROUND: Right hepatic duct, formed by the confluence of the anterior and posterior right sectorial ducts, joins left hepatic duct to form common hepatic duct. This fashion of confluence does not prevail in all cases. The sectorial ducts can aberrantly meet left duct and rest of the ducts from the left lobe of liver. Presence of such variation imposes clinical importance during peri-hilar, split liver transplant surgery or cholecystectomy. Nepalese population has not been explored before disregarding clinical necessity as MRI or cholangiography. METHODS: Descriptive cross sectional study was conducted in 107 cases dissecting the main portal fissure separating hemi liver and extrahepatic biliary confluences. Methylene blue dye was injected and bile duct wall was cut open to the study pattern of the confluence. Data analysis was done with Statistical Package for Social Sciences (SPSS) version 17. RESULTS: Normal variant of confluence was found in 72% cases, aberrant right posterior sectorial duct joins left hepatic duct in 9.3% and aberrant right anterior duct or low insertion of the right posterior sectorial duct was found in 1.9%. 9.3% of cases there is no true right hepatic duct often described as triple confluence. 0.9% cases showed no particular pattern of confluence where common hepatic duct is formed by multiple confluence. Quadrate lobe was found to be draining into right anterior sectorial duct in a single case. CONCLUSIONS: Right hepatic duct confluence pattern is variable and all the evidence occurs at the main portal fissure. Right sectorial duct may join the left duct avoiding normal confluence pattern. Right posterior sectorial duct may be inserted low in the common bile duct.

Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley, Nepal.

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90-100%) with concentrations of 4.5-9.1 and 6.2-7.0 log10 copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30-40%. PMMoV and crAssphage were detected in 40-100% and 8-90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal-Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log10 copies/L) compared with PMMoV (3.3 ± 1.4 log10 copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome.

The protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ∼80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ∼400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.

Reduction of Arcobacter at Two Conventional Wastewater Treatment Plants in Southern Arizona, USA.

This study aimed to identify the bacterial community in two wastewater treatment plants (WWTPs) and to determine the occurrence and reduction of Arcobacter, along with virulence genes (ciaB and pldA). A total of 48 samples (24 influent and 24 effluent) were collected at two WWTPs in southern Arizona in the United States, monthly from August 2011 to July 2012. Bacterial DNA extract was utilized for 16S rRNA metagenomic sequencing. Quantification of Arcobacter 16S rRNA gene was conducted using a recently developed SYBR Green-based quantitative PCR assay. Among 847 genera identified, 113 (13%) were identified as potentially pathogenic bacteria. Arcobacter 16S rRNA gene was detected in all influent samples and ten (83%) and nine (75%) effluent samples at each plant, respectively. Log reduction ratios of Arcobacter 16S rRNA gene in Plant A and Plant B were 1.7 ± 0.9 (n = 10) and 2.3 ± 1.5 (n = 9), respectively. The ciaB gene was detected by quantitative PCR in eleven (92%) and twelve (100%) of 12 influent samples from Plant A and Plant B, respectively, while the pldA gene was detected in eight (67%) and six (50%) influent samples from Plant A and Plant B, respectively. The prevalence of potentially pathogenic bacteria in WWTP effluent indicated the need for disinfection before discharge into the environment.

Co-Infection by Waterborne Enteric Viruses in Children with Gastroenteritis in Nepal.

Enteric viruses are highly contagious and a major cause of waterborne gastroenteritis in children younger than five years of age in developing world. This study examined the prevalence of enteric virus infection in children with gastroenteritis to identify risk factors for co-infections. In total, 107 stool samples were collected from patients with acute gastroenteritis along with samples of their household drinking water and other possible contamination sources, such as food and hand. The presence of major gastroenteritis-causing enteric virus species (group A rotaviruses, enteroviruses, adenoviruses, and noroviruses of genogroup I) in stool and water samples was examined using quantitative polymerase chain reaction. Among the 107 stool samples tested, 103 (96%) samples contained at least one of the four tested enteric viruses, and the combination of group A rotaviruses and enteroviruses was the most common co-infection (52%, n = 54/103). At least one viral agent was detected in 16 (16%) of 103 drinking water samples. Identical enteric viruses were detected in both the stool and water samples taken from the same patients in 13% of cases (n = 13/103). Group A rotaviruses were most frequently found in children suffering from acute diarrhea. No socio-demographic and clinical factors were associated with the risk of co-infection compared with mono-infection. These less commonly diagnosed viral etiological agents in hospitals are highly prevalent in patients with acute gastroenteritis.