Absar Alum

The global war against intestinal parasites—should we use a holistic approach?

Intestinal parasite infections have been known to compromise the quality of human life since prehistoric times. Throughout the ages, human civilizations have fought against parasitism, including intestinal parasites. During the second half of the last century, the focus of disease-combating efforts moved to other imminent public health issues, under the notion that the fight against infectious diseases had been won. However, the disease incidence data indicate that these diseases continue to wreak havoc on human productivity across the globe. Lately, chemotherapeutic intervention has been stressed for the control of intestinal parasitic infections. In this paper we evaluate the need for a holistic approach to address this issue and make recommendations for this cause.

PCR inhibitor levels in concentrates of biosolid samples predicted by a new method based on excitation-emission matrix spectroscopy

ABSTRACT Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with glycine-based methods, PCR inhibitors still persist in concentrations that may interfere with nucleic acid amplification. This results in considerable loss of time and resources and increases the probability of false negatives. A method to estimate the degree of inhibition prior to application of molecular methods is desirable. Here we report fluorescence excitation-emission matrix (EEM) profiling as a tool for predicting levels of molecular inhibition in sample concentrates of biosolids.

Removal of adenovirus, calicivirus, and bacteriophages by conventional drinking water treatment

This study was conducted to evaluate the removal of adenovirus, feline calicivirus (FCV), and bacteriophages MS-2, fr, PRD-1, and Φ X-174 during conventional drinking water treatment using ferric chloride as a coagulant. Adenovirus and FCV were removed to a greater extent than PRD-1 and Φ X-174, indicating that these bacteriophages may be appropriate surrogates for both adenovirus and FCV. Of the four bacteriophages studied in the pilot plant, MS-2 was removed to the greatest extent (5.1 log), followed by fr (4.9 log), PRD-1 (3.5 log), and Φ X-174 (1.3 log). The virus removal trend in the pilot-scale testing was similar to the bench-scale testing; however, the bench-scale testing seemed to provide a conservative estimate of the pilot plant performance. In the pilot-scale testing, MS-2 and fr were removed with the greatest efficiency during filtration, whereas PRD-1 and Φ X-174 showed the greatest removal during sedimentation.

Removal and inactivation of Cryptosporidium and microbial indicators by a quaternary ammonium chloride (QAC)-treated zeolite in pilot filters

A set of pilot filters packed with Zeolite filter media treated with a quaternary ammonium chloride (QAC) were evaluated to verify the proof of concept of their combined antimicrobial capabilities. Escherichia coli was removed and inactivated the most (2.83 log), followed by MS-2 (2.75 log), Klebsiella terriena (2.21 log), PRD-1 (1.95 log), Chlorella vulgaris (1.92 log), and Cryptosporidium parvum oocysts (1.78 log). Especially, inactivation of C. parvum oocysts (1.19 log) was higher than physical removal (0.54 log). The data suggest that QAC-treated Zeolite in the pilot filters has microbial inactivation capabilities and may have useful applications in other types of filter media.

Impact of Environmental Conditions on the Survival of Cryptosporidium and Giardia on Environmental Surfaces

The objective of this study was to find out the impact of environmental conditions on the survival of intestinal parasites on environmental surfaces commonly implicated in the transmission of these parasites. The study was performed by incubating Cryptosporidium and Giardia (oo)cysts on environmentally relevant surfaces such as brushed stainless steel, formica, ceramic, fabric, and skin. Parallel experiments were conducted using clean and soiled coupons incubated under three temperatures. The die-off coefficient rates (K) were calculated using first-order exponential formula. For both parasites, the fastest die-off was recorded on fabric, followed by ceramic, formica, skin, and steel. Die-off rates were directly correlated to the incubation temperatures and surface porosity. The presence of organic matter enhanced the survivability of the resting stages of test parasites. The decay rates calculated in this study can be used in models for public health decision-making process and highlights the mitigation role of hand hygiene agents in their prevention and control.

ECC-RT-PCR: a new method to determine the viability and infectivity of Giardia cysts.

BACKGROUND Giardia sp is a major cause of diarrheal illness worldwide, and millions of people are infected each year. Rapid methods to determine the infectivity and virulence of isolates are critical for the development of intervention strategies to control the transmission of Giardia sp cysts, which occurs through contaminated surfaces, food, and water. However, determining the viability, infectivity, and virulence of Giardia sp cysts using molecular methods is a technical challenge because of the lack of a cell culture model. METHOD This study was designed to evaluate mRNA expression in trophozoites and to assess trophozoite attachment to cell monolayer and changes in transcellular resistance as an indicator of Giardia sp viability and infectivity. Heat shock mRNA in Giardia cysts and variant-specific protein (VSP) mRNA in trophozoites were quantified by reverse transcription polymerase chain reaction (RT-PCR). C2bb (Caco-2) cells were grown on transwell chambers to study the attachment of trophozoites, changes in transcellular resistance, and expression of VSP in trophozoites. RESULTS The results of these molecular and cell culture studies indicate a direct linear correlation between the viability and infectivity of fresh stocks of Giardia sp cysts. The attachment of trophozoites to cell monolayer, expression of VSP, and change in the transcellular resistance was directly correlated with their infectivity in neonatal mice. PCR was successfully combined with the electrophysiological analysis of cell culture (ECC-RT-PCR) post-trophozoite attachment. CONCLUSION This study shows that the ECC-RT-PCR, a new integrated cell culture assay, can be used as a rapid and cost-effective tool for assessing the viability and infectivity of environmental isolates of Giardia sp cysts.

A biosensor platform for rapid detection of E. coli in drinking water.

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.

Comparison of molecular markers for determining the viability and infectivity of Cryptosporidium oocysts and validation of molecular methods against animal infectivity assay

BACKGROUND Globally, disinfectants are widely used to intervene in the dissemination of Cryptosporidium oocysts. However, extensive investigations of oocyst inactivation by various disinfectants are not feasible due to the limitations imposed by animal infectivity methods. Molecular techniques provide an alternative strategy; however, non-metabolic genes have been used as markers for determining viability/infectivity. METHODS In this study we used amyloglucosidase (AG) - a metabolic protein - as a marker to determine viability/infectivity of Cryptosporidium. Oocysts were exposed to 6% hydrogen peroxide for 2min. Samples were analyzed by cell culture polymerase chain reaction (CC-PCR) using PCR primers specific for heat shock protein 70 (hsp70) and AG. Both target genes were amplified with the same level of intensity. RESULTS Based on the results it can be concluded that AG is a valid target for the study of environmental survival and for the evaluation of the efficacy of microbicides against Cryptosporidium using molecular and cellular assays. Comparison of the CC-PCR assay and mouse infectivity assay showed a fairly good correlation under these test conditions. CONCLUSION Results indicate that the CC-PCR assay presents a valid and cost-effective alternative to the mouse infectivity assay.

Cell Surface Display of Poliovirus Receptor on Escherichia coli, a Novel Method for Concentrating Viral Particles in Water

ABSTRACT The lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface of Escherichia coli cells by using the ice nucleation protein (INP) gene. The hPVR gene was ligated to the 3′ end of the INP gene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane of E. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineered E. coli cells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.

Aerobiology of the built environment: Synergy between Legionella and fungi.

BACKGROUND The modern built environment (BE) design creates unique ecological niches ideal for the survival and mutual interaction of microbial communities. This investigation focused on the synergistic relations between Legionella and the fungal species commonly found in BEs and the impact of these synergistic relationships on the survival and transmission of Legionella. METHODS A field study was conducted to identify the types and concentrations of fungi in BEs. The fungal isolates purified from BEs were cocultured with Legionella to study their synergistic association. Cocultured Legionella cells were aerosolized in an air-tight chamber to evaluate the efficacy of ultraviolet (UV) to inactivate these cells. RESULTS Aspergillus, Alternaria, and Cladosporium were the most common fungi detected in samples that tested positive for Legionella. After coculturing, Legionella cells were detected inside fungal hyphae. The microscopic observations of Legionella internalization in fungal hyphae were confirmed by molecular analyses. UV disinfection of the aerosolized Legionella cells that were cocultured with fungi indicated that fungal spores and propagules act as a shield against UV radiation. The shield effect of fungal spores on Legionella cells was quantified at >2.5 log10. CONCLUSIONS This study provides the first evidence, to our knowledge, of Legionella cell presence inside fungi detected in an indoor environment. This symbiotic relationship with fungi results in longer survival of Legionella under ambient conditions and provides protection against UV rays.