Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Surveillance of Naegleria fowleri in Louisiana's public water systems.

The free-living amoeba Naegleria fowleri (Nf) inhabits soil and natural waters worldwide: it is thermophilic and thrives at temperatures up to 45 °C and in a multitude of environments. Three deaths in Louisiana were attributed to primary amoebic meningoencephalitis (PAM) caused by Nf infection in 2011 and 2013. Following these incidents, public water systems are now monitored for the presence of Nf in Louisiana. From 2014 to 2018, 29% (27/93) of samples collected showed positive for Nf and 68% (63/93) showed all thermophilic amoeba culture. Ten raw water sources and 17 distribution water systems tested positive. The year 2017 showed the highest number of samples with Nf (n = 10) followed by nine samples in 2015. As climate change increases surface water temperatures, continued testing for Nf prevalence will be an important facet of water monitoring and will need to extend into locations farther north than the current most common range.

HABs Karenia brevis and Pseudo-nitzschia pre- and post-Hurricane Michael.

Increased occurrences of harmful algal blooms (HAB) in the Gulf of Mexico, and even worldwide, yield concern for increases in brevetoxin exposure leading to respiratory illness or even death, highlighting the need for extensive scientific research and human health monitoring. It is known that major events such as tropical storms and hurricanes are followed by periods of increased red tides caused by HABs; however, the nature by which phytoplankton blooms proliferate following major events remains a topic of great interest and research. The impact of Hurricane Michael on October 10, 2018 on HABs in the Florida panhandle was examined by analyzing data from the Florida Fish and Wildlife Conservation Commission in coordination with Normalized Fluorescence Line Height (nFLH) data from the University of South Florida College of Marine Science. Results presented here demonstrate four phases of HABs during storm events: 1. Pre-storm concentrations, 2. Decreased concentration during the storm, 3. Elevated concentrations following the storm and 4. Recovery period. This time frame can serve to be important in understanding the health dynamics of coastal systems following major storm events.

Classifying Building Roof Damage Using High Resolution Imagery for Disaster Recovery.

Post-hurricane damage assessments are often costly and time-consuming. Remotely sensed data provides a complementary method of data collection that can be completed comparatively quickly and at relatively low cost. This study focuses on 15 Florida counties impacted by Hurricane Michael (2018), which had category 5 strength winds at landfall. The present study evaluates the ability of aerial imagery collected to cost-effectively measure blue tarps on buildings for disaster impact and recovery. A support vector machine model classified blue tarp, and parcels received a damage indicator based on the model's prediction. The model had an overall accuracy of 85.3% with a sensitivity of 74% and a specificity of 96.7%. The model results indicated approximately 7% of all parcels (27 926 residential and 4431 commercial parcels) in the study area as having blue tarp present. The study results may benefit jurisdictions that lacked financial resources to conduct on-the-ground damage assessments.

The importance of accessory protein variants in the pathogenicity of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS- CoV-2) with an estimated fatality rate of less than 1%. The SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 possess putative functions to manipulate host immune mechanisms. These involve interferons, which appear as a consensus function, immune signaling receptor NLRP3 (NLR family pyrin domain-containing 3) inflammasome, and inflammatory cytokines such as interleukin 1ß (IL-1ß) and are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins were observed across six continents of all complete SARS-CoV-2 proteomes based on the data reported before November 2020. A decreasing order of percentage of unique variations in the accessory proteins was determined as ORF3a > ORF8 > ORF7a > ORF6 > ORF10 > ORF7b across all continents. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. These findings suggest that the wide variations in accessory proteins seem to affect the pathogenicity of SARS-CoV-2.

Periodically aperiodic pattern of SARS-CoV-2 mutations underpins the uncertainty of its origin and evolution.

Various lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have contributed to prolongation of the Coronavirus Disease 2019 (COVID-19) pandemic. Several non-synonymous mutations in SARS-CoV-2 proteins have generated multiple SARS-CoV-2 variants. In our previous report, we have shown that an evenly uneven distribution of unique protein variants of SARS-CoV-2 is geo-location or demography-specific. However, the correlation between the demographic transmutability of the SARS-CoV-2 infection and mutations in various proteins remains unknown due to hidden symmetry/asymmetry in the occurrence of mutations. This study tracked how these mutations are emerging in SARS-CoV-2 proteins in six model countries and globally. In a geo-location, considering the mutations having a frequency of detection of at least 500 in each SARS-CoV-2 protein, we studied the country-wise percentage of invariant residues. Our data revealed that since October 2020, highly frequent mutations in SARS-CoV-2 have been observed mostly in the Open Reading Frame (ORF) 7b and ORF8, worldwide. No such highly frequent mutations in any of the SARS-CoV-2 proteins were found in the UK, India, and Brazil, which does not correlate with the degree of transmissibility of the virus in India and Brazil. However, we have found a signature that SARS-CoV-2 proteins were evolving at a higher rate, and considering global data, mutations are detected in the majority of the available amino acid locations. Fractal analysis of each protein's normalized factor time series showed a periodically aperiodic emergence of dominant variants for SARS-CoV-2 protein mutations across different countries. It was noticed that certain high-frequency variants have emerged in the last couple of months, and thus the emerging SARS-CoV-2 strains are expected to contain prevalent mutations in the ORF3a, membrane, and ORF8 proteins. In contrast to other beta-coronaviruses, SARS-CoV-2 variants have rapidly emerged based on demographically dependent mutations. Characterization of the periodically aperiodic nature of the demographic spread of SARS-CoV-2 variants in various countries can contribute to the identification of the origin of SARS-CoV-2.

Remotely sensed measures of Hurricane Michael damage and adverse perinatal outcomes and access to prenatal care services in the Florida panhandle.

BACKGROUND: Studies of effects of hurricanes on perinatal outcomes often rely on approximate measures of exposure. This study aims to use observed damage from aerial imagery to refine residential building damage estimates, evaluate the population changes post landfall, and assess the associations between the extent of residential building damage and adverse perinatal outcomes and access to prenatal care (PNC) services.  METHODS: Vital statistics data from the Florida Department of Health's Office of Vital Statistics were used to align maternal geocoded address data to high-resolution imagery (0.5-foot resolution, true color with red, blue, and green bands) aerial photographs. Machine learning (support vector machines) classified residential roof damage across the study area. Perinatal outcomes were compared with the presence or absence of damage to the mother's home. Log-binomial regression models were used to compare the populations living in and outside of high-risk/damage areas, to assess the population changes after Hurricane Michael, and to estimate the associations between damage after Hurricane Michael and adverse perinatal outcomes/access to PNC services. A semi-parametric linear model was used to model time of first PNC visit and increase in damage. RESULTS: We included 8,965 women in analysis. Women with lower education and/or of Black or other non-White race/ethnicity were more likely to live in areas that would see high damage than other groups. Moreover, there was a greater proportion of births delivered by women living in the high-risk/damage area (> 25% damaged parcels after Michael) in the year before Michael than the year after Michael. Lastly, living in the area with relatively high damage increased the risk of having intermediate or inadequate PNC (adjusted Risk Ratio = 1.21, 95% CI: 1.03, 1.43), but not other adverse perinatal outcomes. CONCLUSIONS: Aerially observed damage data enable us to evaluate the impact of natural disasters on perinatal outcomes and access to PNC services based on residential building damage immediately surrounding a household. The association between the extent of damage and adverse perinatal outcomes should be further investigated in future studies.

Microbial source tracking of fecal contamination in stormwater runoff.

Concerns over fecal contamination in stormwater canals have promoted the need for pollution control strategies, including the use of microbial source tracking, to identify fecal contamination in the Greater New Orleans Area. Surface water samples were collected over a 12-month period at five canal locations within Jefferson Parish, Louisiana. Quantitative polymerase chain reaction and the IDEXX method were used to assess the concentrations of coliforms, Escherichia coli (E. coli) and human fecal 183 bacteroides (HF183) in stormwater samples. A 100% positive detection rate of total coliforms and E. coli was observed across all tested sites. Despite the closeness of the five sites, when averaged across all sampling time points, Kruskal-Wallis tests indicated that E. coli was present at significantly different concentrations in these locations (χ2(5) = 19.8, p = 0.0005). HF183 was detected in 62% of the water samples collected during the stormwater sampling. Without further testing for HF183 markers, the conclusion from this study would have been that fecal contamination from an unknown source was always present at varying levels during the study period. Analysis of HF183 markers therefore adds another layer of conclusions to the results deductible from E. coli concentrations. A 100% E. coli detection rate, high E. coli concentrations coupled with low rates of HF183 detection particularly at the Esplanade, Poplar Street, and Bonnabel Boat Launch sites, the sites closest to the lake outlet, throughout the study period, indicate that fecal contamination at these stormwater canal sites comes primarily from non-human sources. However, the Metairie Road and Napoleon Avenue sites, which have the highest HF183 detection rates, on top of chronic pollution by other non-human sources, are also influenced by human fecal pollution, possibly because of human development and faulty infrastructure. This study highlights the advantages of the use of microbial source-tracking methods to complement traditional indicator bacteria.

Occurrence of Naegleria fowleri and faecal indicators in sediments from Lake Pontchartrain, Louisiana.

The occurrence of amoeba, Naegleria fowleri, in sediment samples from Lake Pontchartrain in Louisiana was investigated. This amoeba is pathogenic and can cause primary amoebic meningoencephalitis. In this study, quantitative polymerase chain reaction methods were used to test for the prevalence of Naegleria fowleri, HF183, and E. coli. N. fowleri was detected in 51.25% of our sediment samples. Illumina sequencing of sediment samples revealed ten different phyla, with Cyanobacteria being the most predominant at sites that generally presented with the highest median N. fowleri concentrations. N. fowleri was however strongly negatively correlated with HF183 (r = -0.859, p < 0.001). Whenever sediment E. coli concentrations were below 1.54 Log GC/g, there was only a 37.5% chance that N. fowleri would be detected in the same sample. When sediment E. coli concentrations exceeded 2.77 Log GC/g, the chances of detecting N. fowleri in the same sample increased to 90%, potentially suggesting predatory activity by the amoeba. The effect of temperature was observed to be different in relation to observed N. fowleri concentrations and detection rates. Although sediment samples collected during periods of higher temperatures had significantly lower mean N. fowleri concentrations (2.7 Log GC/g) compared to those collected at lower temperatures (3.7 Log GC/g, t(39) = 4.167, p < 0.001), higher N. fowleri detection rates in the overall samples were observed at higher temperatures (>19.1 °C) than at lower temperatures (<19.1 °C).

Harmful algal bloom-related 311 calls, Cape Coral, Florida 2018-2019.

Harmful algal blooms (HABs) can adversely impact water quality and threaten human and animal health. People working or living along waterways with prolonged HAB contamination may face elevated toxin exposures and breathing complications. Monitoring HABs and potential adverse human health effects is notoriously difficult due to routes and levels of exposure that vary widely across time and space. This study examines the utility of 311 calls to enhance HAB surveillance and monitoring. The study focuses on Cape Coral, FL, USA, located along the banks of the Caloosahatchee River and Estuary and the Gulf of Mexico. The wider study area experienced a prolonged cyanobacteria bloom in 2018. The present study examines the relationship between weekly water quality characteristics (temperature, dissolved oxygen, pH, microcystin-LR) and municipal requests for information or services (algal 311 calls). Each 1 µg/L increase in waterborne microcystin-LR concentrations corresponded with 9% more algal 311 calls (95% confidence interval: 1.03-1.15, p = 0.002). The results suggest water quality monitoring and the 311 dispatch systems may be further integrated to improve public health surveillance.

Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: Implications for wastewater-based epidemiology.

We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.

Molecular detection of opportunistic premise plumbing pathogens in rural Louisiana's drinking water distribution system.

Opportunistic premise plumbing pathogens (OPPPs) in drinking water distribution systems are responsible for causing numerous infections such as Legionnaires' disease and pneumonia through the consumption of contaminated drinking water. The incidence of opportunistic pathogens and the number of individuals at risk of contracting infections caused by these OPPPs in drinking water has risen drastically in the past decade. Preflush and postflush water samples were collected from 64 houses in a rural town in northeast Louisiana to determine drinking water quality in terms of understanding abiotic and biotic factors on potential proliferation of OPPPs. Physical and chemical water quality parameters, such as pH, temperature, dissolved oxygen, salinity, and specific conductance were also measured. The quantitative polymerase chain reaction (qPCR) results indicated that Legionella spp. had the highest prevalence and was found in 46/64 samples (72%), followed by Mycobacterium spp. which was found in 43/64 samples (67%), E. coli in 31/64 samples (48%) and, Naegleria fowleri in 4/64 samples (6%) respectively. The results indicate the persistence of Legionella spp. DNA marker in these water samples.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Molecular detection of opportunistic pathogens and insights into microbial diversity in private well water and premise plumbing.

Private well water systems in rural areas that are improperly maintained will result in poor drinking water quality, loss of water supply, and pose human health risk. The purpose of this study was to investigate the occurrence of fecal indicator bacteria (FIB) and opportunistic pathogens in private well water in rural areas surrounding New Orleans, Louisiana. Our results confirmed the ubiquitous nature of Legionella (86.7%) and mycobacteria (68.1%) in private well water in the study area, with gene concentration ranged from 0.60 to 5.53 and 0.67 to 5.95 Log10 of GC/100 mL, respectively. Naegleria fowleri target sequence was detected in 16.8% and Escherichia coli was detected in 43.4% of the water samples. Total coliform, as well as Legionella and mycobacteria genetic markers' concentrations were significantly reduced by 3-minute flushing. Next-generation sequencing (NGS) data indicated that the abundance of bacterial species was significantly increased in water collected in kitchens compared with samples from wells directly. This study provided integrated knowledge on the persistence of pathogenic organisms in private well water. Further study is needed to explore the presence of clinical species of those opportunistic pathogens in private well water systems to elucidate the health risk.

Possible Transmission Flow of SARS-CoV-2 Based on ACE2 Features.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment.

Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates; however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and viruses (pepper mild mottle virus, Aichi virus 1, adenovirus, and polyomaviruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters.

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.

Near Real-Time Detection of E. coli in Reclaimed Water.

Advanced treatment of reclaimed water prior to potable reuse normally results in the inactivation of bacterial populations, however, incremental treatment failure can result in bacteria, including pathogens, remaining viable. Therefore, potential microorganisms need to be detected in real-time to preclude potential adverse human health effects. Real-time detection of microbes presents unique problems which are dependent on the water quality of the test water, including parameters such as particulate content and turbidity, and natural organic matter content. In addition, microbes are unusual in that: (i) viability and culturability are not always synonymous; (ii) viability in water can be reduced by osmotic stress; and (iii) bacteria can invoke repair mechanisms in response to UV disinfection resulting in regrowth of bacterial populations. All these issues related to bacteria affect the efficacy of real-time detection for bacteria. Here we evaluate three different sensors suitable for specific water qualities. The sensor A is an on-line, real-time sensor that allows for the continuous monitoring of particulates (including microbial contaminants) using multi-angle-light scattering (MALS) technology. The sensor B is a microbial detection system that uses optical technique, Mie light scattering, for particle sizing and fluorescence emission for viable bacteria detection. The last sensor C was based on adenosine triphosphate (ATP) production. E. coli was used a model organism and out of all tested sensors, we found the sensor C to be the most accurate. It has a great potential as a surrogate parameter for microbial loads in test waters and be useful for process control in treatment trains.