Characterization of red blood cells with multiwavelength transmission spectroscopy.

Multiwavelength transmission (MWT) spectroscopy was applied to the investigation of the morphological parameters and composition of red blood cells (RBCs). The MWT spectra were quantitatively analyzed with a Mie theory based interpretation model modified to incorporate the effects of the nonsphericity and orientation of RBCs. The MWT spectra of the healthy and anemic samples were investigated for the RBC indices in open and blinded studies. When MWT performance was evaluated against a standard reference system, very good agreement between two methods, with R (2) > 0.85 for all indices studied, was demonstrated. The RBC morphological parameters were used to characterize three types of anemia and to draw an association between RBC morphology and anemia severity. The MWT spectra of RBCs infected with malaria parasite Plasmodium falciparum at different life cycle stages were analyzed for RBC morphological parameters. The changes in the RBC volume, surface area, aspect ratio, and hemoglobin composition were used to trace the morphological and compositional alterations in the infected RBCs occurring with parasites' development and to provide insights into parasite-host interactions. The MWT method was shown to be reliable for determination of the RBC morphological parameters and to be valuable for identification of the RBC pathologic changes and disease states.

Spectrophotometric detection of susceptibility to anti-malarial drugs.

BACKGROUND: With malaria drug resistance increasing in prevalence and severity, new technologies are needed to aid and improve the accuracy and clinical relevance of laboratory or field testing for malaria drug resistance. This study presents a method based on simple and reagentless spectroscopic measurements coupled with comprehensive spectral interpretation analysis that provides valuable quantitative information on the morphological and compositional responses of Plasmodium falciparum and infected red blood cells (IRBCs) to anti-malarial treatment. METHODS: The changes in the size, internal structure, nucleotide and haemozoin composition of the parasites as well as the morphology (size and shape) and haemoglobin composition of the IRBCs treated with dihydroartemisinin (DHA) and mefloquine (MFQ) were investigated using a spectral interpretation analysis. RESULTS: DHA treatment reduced the sizes of the parasites and their structural organelles. The haemoglobin composition of the host IRBCs determined from spectroscopic analysis changed negligibly following DHA treatment. MFQ treated parasites grew to the same size as those from parallel non-treated cultures but lacked haemozoin. Lesser deformation of the cell shape and no haemoglobin depletion were detected for the IRBCs of MFQ treated cultures. CONCLUSIONS: The spectroscopic analysis method proved to be sensitive for recognition of the effects of anti-malarial treatment on the structure and composition of the parasites and IRBCs. The method can have significant potential for research and clinical applications such as evaluating patient specimens for drug action, drug effects or for therapeutic monitoring.

Multiwavelength transmission spectroscopy revisited for the characterization of the protein and polystyrene nanoparticle mixtures.

Multiwavelength Transmission (MWT) UV-Vis-NIR spectroscopy, an effective technique often underutilized for the characterization of processes involving particulates, such as protein aggregation, is systematically explored using bovine serum albumin and a set of NIST-traceable particle size (PS) standards having certified particle diameters over the nominal size range of 30 to 100 nm. The PS standards are used as surrogates for protein aggregates and other contaminants such as oils and microbubbles. Therefore, the standards can be used to quantitatively modify the optical properties of protein solutions and thus observe the effect of the presence of aggregates and other particulates on their wavelength-dependent transmission spectra. The experimental results demonstrate that the changes induced in the optical density spectra of proteins due to the presence of PS particles are detectable and consistent with the expectations set by light scattering theory. It is demonstrated that the size and relative concentrations of the particle populations present in the protein samples can be quantified. Because of the considerable dynamic range of MWT UV-Vis-NIR spectroscopy for particle analysis and its real-time measurement capabilities, this type of spectroscopy can be effectively used for the characterization of protein aggregates and for the continuous real-time monitoring of aggregation processes and for the identification and quantification of contaminants in protein-based products.

Multiwavelength transmission spectroscopy revisited for the characterization of the protein and polystyrene nanoparticle interactions.

Multiwavelength transmission ultraviolet-visible-near-infrared (UV-Vis-NIR) spectroscopy is an effective technique that has not yet been fully exploited for the characterization of products of protein and particle interactions. Here, it is explored by using bovine serum albumin and National Institute of Standards and Technology-traceable particle size standard having a nominal diameter of 20 nm. Adsorption of bovine serum albumin to the particles is quantitatively ascertained through its effect on the wavelength-dependent transmission spectra of protein and particle mixtures. The experimental results demonstrate that the changes induced in the transmission spectra of protein and particle mixtures because of protein adsorption on particles are detectable and consistent with the expectations set by the light-scattering theory. The size, structure, composition, and relative concentrations of the particle populations present in the protein-particle mixtures can be quantified. Given the considerable dynamic range of multiwavelength transmission UV-Vis-NIR spectroscopy for particle analysis and its real-time measurement capabilities, this type of spectroscopy can be effectively used for the characterization of the products of protein-particle interaction and for the continuous real-time monitoring of interaction processes.

Reagent-free bacterial identification using multivariate analysis of transmission spectra.

The identification of bacterial pathogens from culture is critical to the proper administration of antibiotics and patient treatment. Many of the tests currently used in the clinical microbiology laboratory for bacterial identification today can be highly sensitive and specific; however, they have the additional burdens of complexity, cost, and the need for specialized reagents. We present an innovative, reagent-free method for the identification of pathogens from culture. A clinical study has been initiated to evaluate the sensitivity and specificity of this approach. Multiwavelength transmission spectra were generated from a set of clinical isolates including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Spectra of an initial training set of these target organisms were used to create identification models representing the spectral variability of each species using multivariate statistical techniques. Next, the spectra of the blinded isolates of targeted species were identified using the model achieving >94% sensitivity and >98% specificity, with 100% accuracy for P. aeruginosa and S. aureus. The results from this on-going clinical study indicate this approach is a powerful and exciting technique for identification of pathogens. The menu of models is being expanded to include other bacterial genera and species of clinical significance.

Multi-wavelength transmission spectroscopy revisited for micron and submicron particle characterization.

Multi-wavelength transmission (MWT) ultraviolet-visible-near-infrared (UV-Vis-NIR) spectroscopy, a technique underappreciated for particle characterization, is systematically explored using a set of NIST traceable standards over the nominal size range of 20 to 20,000 nm. Experimental results demonstrate that the particle size distributions obtained from MWT spectral data are in excellent agreement with the values reported by the manufacturer. In addition, it is shown that quantitative information on the particle concentration can be obtained--which is not currently accessible from commercially available light scattering instrumentation. The results validate that MWT UV-Vis-NIR spectroscopy has a considerable dynamic range for particle size measurements and offers significant advantages over other particle characterization techniques. Among these are the simplicity of the instrumentation and the measurements and the wealth of quantitative information contained in the MWT spectra. Most importantly, with standardized measurement protocols and standardized spectrometer configurations, MWT measurements can be used to provide the user and the manufacturer of particles with traceable data (i.e., the spectra and the quantitative analysis) for quality assurance.

New method for the detection of micro-organisms in blood: application of quantitative interpretation model to aerobic blood cultures.

The physical and chemical changes occurring in blood that has been inoculated into a blood culture bottle can be used as means to detect the presence of microorganisms in blood cultures. These changes include primarily the conversion of oxy- to deoxyhemoglobin within the red blood cells (RBCs) and changes in the cell number densities. These changes in the physical and chemical properties of blood can be readily detected using spectrophometric methods thus enabling the continuous monitoring of blood culture vials to provide quantitative information on the growth behavior of the microorganisms present. This paper reports on the application of spectrophotometric information obtained from diffuse reflectance measurements of aerobic blood cultures to detect microbial growth and compares the results to those obtained using the standard blood culture system.

Quantitative interpretations of Visible-NIR reflectance spectra of blood.

This paper illustrates the implementation of a new theoretical model for rapid quantitative analysis of the Vis-NIR diffuse reflectance spectra of blood cultures. This new model is based on the photon diffusion theory and Mie scattering theory that have been formulated to account for multiple scattering populations and absorptive components. This study stresses the significance of the thorough solution of the scattering and absorption problem in order to accurately resolve for optically relevant parameters of blood culture components. With advantages of being calibration-free and computationally fast, the new model has two basic requirements. First, wavelength-dependent refractive indices of the basic chemical constituents of blood culture components are needed. Second, multi-wavelength measurements or at least the measurements of characteristic wavelengths equal to the degrees of freedom, i.e. number of optically relevant parameters, of blood culture system are required. The blood culture analysis model was tested with a large number of diffuse reflectance spectra of blood culture samples characterized by an extensive range of the relevant parameters.

Effects of wastewater disinfection on waterborne bacteria and viruses.

Wastewater disinfection is practiced with the goal of reducing risks of human exposure to pathogenic microorganisms. In most circumstances, the efficacy of a wastewater disinfection process is regulated and monitored based on measurements of the responses of indicator bacteria. However, inactivation of indicator bacteria does not guarantee an acceptable degree of inactivation among other waterborne microorganisms (e.g., microbial pathogens). Undisinfected effluent samples from several municipal wastewater treatment facilities were collected for analysis. Facilities were selected to provide a broad spectrum of effluent quality, particularly as related to nitrogenous compounds. Samples were subjected to bench-scale chlorination and dechlorination and UV irradiation under conditions that allowed compliance with relevant discharge regulations and such that disinfectant exposures could be accurately quantified. Disinfected samples were subjected to a battery of assays to assess the immediate and long-term effects of wastewater disinfection on waterborne bacteria and viruses. In general, (viable) bacterial populations showed an immediate decline as a result of disinfectant exposure; however, incubation of disinfected samples under conditions that were designed to mimic the conditions in a receiving stream resulted in substantial recovery of the total bacterial community. The bacterial groups that are commonly used as indicators do not provide an accurate representation of the response of the bacterial community to disinfectant exposure and subsequent recovery in the environment. UV irradiation and chlorination/dechlorination both accomplished measurable inactivation of indigenous phage; however, the extent of inactivation was fairly modest under the conditions of disinfection used in this study. UV irradiation was consistently more effective as a virucide than chlorination/dechlorination under the conditions of application, based on measurements of virus (phage) diversity and concentration. Taken together, and when considered in conjunction with previously published research, the results of these experiments illustrate several important limitations of common disinfection processes as applied in the treatment of municipal wastewaters. In general, it is not clear that conventional disinfection processes, as commonly implemented, are effective for control of the risks of disease transmission, particularly those associated with viral pathogens. Microbial quality in receiving streams may not be substantially improved by the application of these disinfection processes; under some circumstances, an argument can be made that disinfection may actually yield a decrease in effluent and receiving water quality. Decisions regarding the need for effluent disinfection must account for site-specific characteristics, but it is not clear that disinfection of municipal wastewater effluents is necessary or beneficial for all facilities. When direct human contact or ingestion of municipal wastewater effluents is likely, disinfection may be necessary. Under these circumstances, UV irradiation appears to be superior to chlorination in terms of microbial quality and chemistry and toxicology. This advantage is particularly evident in effluents that contain appreciable quantities of ammonia-nitrogen or organic nitrogen.

Detection of Cryptosporidium parvum oocysts in sediment and biosolids by immunomagnetic separation.

A method for the detection of Cryptosporidium parvum oocysts in sediment and wastewater biosolids has been developed using immunomagnetic separation kits that were designed for use with water. This method requires no pretreatment of the sediment or biosolids samples before the commercial kit application. Oocyst recovery efficiencies from sediment and biosolids using the modified Dynal (Lake Success, New York) and Crypto-Scan commercial methods (Immucell Corporation, Portland, Maine) ranged from 20 to 60%. While the sensitivity of the method is dependent on the amount of sediment processed and the equivalent volume examined under the microscope, it was able to detect 0.48 oocysts per gram dry weight sediment. Using this method, Cryptosporidium parvum oocysts were found at levels as high as 97 oocysts/g of primary biosolids and at levels up to 4 oocysts/g in polluted sediment.

Cryptosporidium parvum: treatment effects and the rate of decline in oocyst infectivity.

Cryptosporidium parvum has become the focus of numerous studies on waterborne disease and transmission in response to outbreaks endangering populations worldwide. The Foci Detection Method-Most Probable Number Assay (FDM-MPN) is an in vitro cell culture method that has been developed and used to determine the quantity of infectious C. parvum oocysts. This research evaluated 2 vendor's producing oocysts, Sterling Parasitology Laboratory (SPL) and Pleasant Hill Farms (PHF) (now known as Bunch Grass Farms as of 12/03), classified as young (<30 days) and aged (>165 days), for comparison of treatments (bleach, antibiotic, no treatment) before cell culture, as well as an age study, to determine any lot-to-lot differences and vendor differences regarding the rate of decline in infectivity. Bleach treatment (0.525%) appeared to be the optimum method for the FDM-MPN with regards to maximum infectivity, efficient disinfection, with no visible antagonistic affects on the C. parvum oocysts. The age study revealed that lot-to-lot variability within each vendor stayed within 1 log10 difference, while the rates of decline in infectivity measured until 107 and 120 days of age when stored at 4 C for SPL and PHF were -0.016 and -0.014 log10 infectious oocysts/day, respectively. These results provide insight regarding C. parvum oocyst viability in a fecal population, as well as useful knowledge for further methods development.

Detection of infectious parasites in reclaimed water.

The presence of infectious protozoan pathogens in reclaimed water may present an unacceptable health risk. This study was designed similar to a study reported by Garcia et al. (2002), which detected no infectious Giardia cysts in the final effluent of a tertiary treatment facility as determined by animal infectivity (dose 1000 cysts/gerbil). This study also included evaluation of Cryptosporidium oocyst infectivity. Infectious Giardia cysts were detected in the final effluent with 1 gerbil out of 3 inoculated with 250 cysts from reclaimed water showing signs of infection 15 days postinoculation. None of the Cryptosporidium oocysts concentrated from the reclaimed water samples appeared to be infectious.

Infectious Cryptosporidium parvum oocysts in final reclaimed effluent.

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection.

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.

Low- and medium-pressure UV inactivation of microsporidia Encephalitozoon intestinalis.

Newly recognized waterborne pathogens such as microsporidia are being detected in the world's water supplies with increasing frequency. Many of these organisms have been shown to cause negative health impacts for both immunocompetent as well as immunocompromised individuals. It is imperative that these emerging pathogens be investigated for their ability to resist both traditional and novel disinfection technologies that are currently in use or under consideration for drinking water treatment. Low- and medium pressure UV light is at the cutting edge of disinfection technologies for the drinking water industry. While previous UV disinfection studies have focused on the inactivation of Cryptosporidium and Giardia as well as viruses and common bacteria, this research reports the ability of low- and medium pressure UV light to inactivate > 3.6 log10 of microsporidia Encephalitozoon intestinalis spores at a dose of 6 mJ/cm2 or higher as determined using a cell culture approach.

Risk and control of waterborne cryptosporidiosis.

Cryptosporidium remains at the forefront of studies on waterborne disease transmission and abatement. The impact of environmental land use patterns which contribute animal and human waste, climatic precipitation leading to a strong association with outbreaks, and community infrastructure and water treatment are now recognized as contributing factors in the potential for waterborne spread of the protozoan. Advances in detection methodologies, including the ability to genotype various strains of this organism, have shown that human wastes are often the source of the contamination and cell culture techniques have allowed insight into the viability of the oocyst populations. Currently water treatment has focused on UV and ozone disinfection as most promising for the inactivation of this protozoan pathogen.