Grantee Research Project Results
Final Report: Noncontact, Optical Molecular Method for Detection and Identification of Cryptosporidium parvum Oocysts in Drinking Water
EPA Contract Number: EPD04032Title: Noncontact, Optical Molecular Method for Detection and Identification of Cryptosporidium parvum Oocysts in Drinking Water
Investigators: Stewart, Shona , Maier, John
Small Business: ChemImage Corporation
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2004 through August 31, 2004
Project Amount: $69,978
RFA: Small Business Innovation Research (SBIR) - Phase I (2004) RFA Text | Recipients Lists
Research Category: Drinking Water , SBIR - Water and Wastewater , Small Business Innovation Research (SBIR)
Description:
Quality standards designed to ensure a safe drinking water supply require a broad array of screening tests for chemicals, environmental contaminants, and microbiological organisms that represent a threat to human health. A particular focus in these screening efforts is Cryptosporidium parvum, a parasite that is responsible for some of the largest outbreaks of waterborne disease in U.S. history. Current testing for C. parvum is performed using fluorescent labeling of oocysts concentrated from water collected in treatment plants. The current methods have several limitations, including dependence on sophisticated chemical reagents (fluorescent-labeled antibodies) for the detection and identification of the oocysts. Nonspecific chemical binding, or poor binding affinity of these complex reagents, can compromise the effectiveness of these techniques. Moreover, the use of these reagents is a significant barrier to more automated screening. These limitations represent an opportunity to develop, validate, and deploy a reagentless method that can detect and identify oocysts in water.
C. parvum is now recognized as one of the principal contributors to drinking water contamination, and is known to have been the cause of several major outbreaks in the past 10 years (Zuckerman, et al., 1999). The largest cryptosporidiosis outbreak in U.S. history occurred in 1993, affecting more than 400,000 people in Milwaukee, WI, and killing more than 50. A major contributor to the size of the outbreak was the initial misdiagnosis of the infection. It took 2 weeks to identify Cryptosporidium as the cause of the outbreak (Morgan and Thompson, 1998; Morbidity and Mortality Weekly Report, 1996).
Cryptosporidium is a protozoan microorganism that causes diarrhea and is not sensitive to antibiotics. In immunocompromised patients, gastrointestinal infections with C. parvum cysts can be life threatening. Infection by this parasite has been documented with ingestion of only a few oocysts, which has led to a zero tolerance level as a goal for treatment of drinking water for this organism. C. parvum detection methods currently in use in the water supply industry typically involve microscopic examination of samples stained with fluorescent antibodies for the presence of cysts. These methods require highly trained microscopists, are time consuming, and often lack sensitivity (Morgan and Thompson, 1998). Variability among antigens of Cryptosporidium isolates can result in lack of detection of some organisms (Griffin, et al., 1992). Similarly, other techniques, which depend on antibody-based labeling such as flow cytometry, also are limited by the specificity and binding affinity of the antibody used in the test. Polymerase chain reaction assays have demonstrated sensitivity to pathogen detection in clinical applications; however, these procedures are expensive and require experienced scientists to run (Bialek, et al., 2002). Additionally, the infectivity or viability of an oocyst cannot be determined using the current technology. The capability of measuring this property would open the door to a new consideration for Cryptosporidium treatment.
Raman spectroscopy and imaging is a molecular diagnostic technique that has demonstrated promise for detection of pathogens (Treado, et al., 2003). Raman spectroscopy uses light scattered from a sample under laser illumination to characterize the sample in terms of molecular composition. Although Raman spectroscopy is not widely used in the field of water contaminant detection, it is an established analytical technique employed commercially in the food, pharmaceutical, and polymer industries. Raman imaging adds a spatial dimension of information, enabling morphological evaluation of the molecular distributions of specific chemicals in complex samples. Raman spectroscopy and imaging do not require specific reagents for identification of microbial species; these techniques rely on the inherent specificity of Raman spectroscopy coupled to the morphometric information accessible through digital imaging. ChemImage Corporation, working in conjunction with collaborators at the Armed Forces Institute of Pathology, the U.S. Army Edgewood Chemical Biological Center, the U.S. Naval Research Laboratory, the U.S. Army Research Laboratory, and other federal organizations have validated Raman spectroscopy and imaging, through blind trials, as a microbial identification tool (Vanni, 2004). Because of its established history of use in analytical chemistry, Raman spectroscopy is being shown to be useful for the concurrent evaluation of water samples for chemicals and microorganisms. This molecular technique offers many advantages over the currently employed methods for detection of Cryptosporidium in water, including sensitivity, specificity, and cost effectiveness. This Phase I research project demonstrated that C. parvum oocysts have a unique and reproducible Raman spectral signature. The goal of this research project was to perform rigorous scientific investigation of this question to lay the groundwork for Phase II.
Summary/Accomplishments (Outputs/Outcomes):
An investigation of sampling techniques and different substrates revealed that Raman spectra of oocysts can be optimized by allowing centrifuged oocysts to dry on aluminum-coated substrates. Acquisition parameters for dispersive spectra were optimized to enable identification-quality spectra to be collected in approximately 2 minutes.
In approximately 2,000 oocysts from six batches procured from two independent sources of C. parvum, the Raman spectra were reproducible. This was determined using standard statistical metrics. The spectrum of the oocysts also can be distinguished from the Raman spectrum of other microorganisms, both vegetative and spores. Library searches of several random spectra identified them amongst 300 Raman spectra of biological and chemical compounds to correspond to C. parvum oocysts. In addition, an estimated Receiver Operating Characteristic curve was constructed to illustrate the very high sensitivity and specificity of Raman spectroscopy for identifying C. oocysts amongst river water components. Optimization of Raman chemical imaging of oocysts in river water exhibited high-quality Raman images over the fingerprint region of the spectrum in approximately 20 minutes. In this stage, a method for conducting Raman chemical imaging on oocysts was optimized and documented, after which mixtures of river water and oocysts were successfully imaged. Even a single oocyst was detected using Raman chemical imaging. Identification of a single oocyst was not as easily achieved using the image spectrum; however, the dispersive spectrum taken of the single oocyst in river water was identified as a C. parvum oocyst with a 92 percent match based on Euclidian Distance metrics.
The results support the hypothesis that Raman spectroscopy and imaging are potentially valuable techniques for the identification of oocysts in river and drinking water.
Conclusions:
This proof-of-concept work showed that ChemImage Corporation’s Raman spectroscopy and imaging technology has application in the water monitoring industry. Not only do the Raman techniques described have the sensitivity to detect single Cryptosporidium oocysts, but they also are a selective enough technique to yield a Raman spectrum characteristic of Cryptosporidium, which can be identified and distinguished from other microorganisms, spores, and inorganic components typically found in river water. This can be achieved at the single oocyst level without the use of reagents. Identification of Cryptosporidium at the single oocyst level is essential for water monitoring, as the acceptable level in drinking water for this organism is zero, according to the U.S. Environmental Protection Agency (EPA).
Sample preparation is simple and fast, and good quality Raman spectra, which identify Cryptosporidium oocysts, can be achieved in approximately 2 minutes. Extensive investigation of several different batches of C. parvum oocysts, such as the Iowa isolate, suggested that the spectrum of this strain is consistent, so much so that spectra chosen at random from each batch were matched with the library spectrum of C. parvum, Iowa isolate. This provided evidence that the Raman technique could be used for identification of the microorganisms. Currently employed methods take much longer to identify Cryptosporidium.
In addition, this Phase I research project demonstrated that ChemImage Corporation’s Raman chemical imaging techniques can be employed to detect and identify C. parvum oocysts in the presence of typical water interferents. Raman imaging has the advantage of collecting spectra simultaneously at every point in a field of view. In the presence of other microorganisms, particles, and perhaps other species of Cryptosporidium oocysts, Raman chemical imaging and the subsequent image processing techniques are able to eliminate the interfering factors to highlight the signal that matches C. parvum oocysts, and from where in the field of view this signal originates.
Currently, oocysts are imaged over the fingerprint region in 20 minutes. ChemImage Corporation anticipates reducing this acquisition time with refinement of the instrumentation and further investigation into more advanced sampling techniques.
The EPA requires mandated screening and testing for Cryptosporidium to be conducted by all water suppliers with more than 10,000 customers. This mandate creates a need for accurate, affordable screening technology by these potential customers. Because of the broad applicability of Raman chemical imaging, it is anticipated that a system capable of screening for Cryptosporidium would be multitasked to achieve other endpoints of interest to water suppliers.
In the long term, as the methods to miniaturize and mass produce this technology are developed, the customer base will expand to smaller water supply systems and portable assessment teams. With appropriately networked systems, a remote water-monitoring network is feasible.
ChemImage Corporation has patents pending that cover the application of Raman chemical imaging for the investigation of microbial samples.
References
Zuckerman U, Armon R, Tzipori S, Gold D. Evaluation of a portable differential continuous flow centrifuge for concentration of Cryptosporidium oocysts and Giardia cysts from water. Journal of Applied Microbiology 1999;86:955.
Morgan UM, Thompson RCA. Molecular detection of parasitic protozoa. Parasitology 1998;117:S73-S85.
Morbidity and Mortality Weekly Report 1996;45(SS-1):1.
Griffin K, Matthai E, Hommel M, Weitz JC, Baxby D, Hart CA. Antigenic diversity among oocysts of clinical isolates of Cryptosporidium parvum. Journal of Protozoology Research 1992;2:97.
Bialek R, Binder N, Dietz KJA, Knobloch J, Zelck UE. Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvum in fecal specimens. Diagnostic Microbiology and Infectious Disease 2002;43:283.
Treado PJ, Vanni GS, Schweitzer B, Nelson MP, Gardner C, Wolfe J, Neiss J, Hadfield T, Samuels A. Chemical imaging for rapid reagentless biothreat detection. Optical Society of America: Optical Sensing for Homeland Security, Washington, DC, February 2003.
Vanni VG. Falcon Blind Trial Report: Raman Chemical Imaging Biothreat Detection (RCIBD) Program. Database Development and Rapid Deployment of Instantaneous Anthrax Detection Technology (Contract # DAMA17-03-C-0091), June 2004.
Supplemental Keywords:
Cryptosporidium parvum , oocyst, waterborne disease, drinking water, drinking water monitoring, detection method, water contamination, cryptosporidiosis, parasite, Raman spectroscopy, SBIR,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Water, Environmental Chemistry, Environmental Monitoring, Drinking Water, Environmental Engineering, cryptosporidium parvum oocysts, monitoring, pathogens, detection, Raman spectroscopy, community water system, cryptosporidium , other - risk managementThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.