The Infectivity ofCryptosporidium parvumin Healthy Volunteers
Herbert L. DuPontCynthia L. ChappellCharles R. SterlingPablo C. OkhuysenJoan B. RoseWalter Jakubowski
894
Citation
34
Reference
10
Related Paper
Citation Trend
Abstract:
Small numbers of Cryptosporidium parvum oocysts can contaminate even treated drinking water, and ingestion of oocysts can cause diarrheal disease in normal as well as immunocompromised hosts. Since the number of organisms necessary to cause infection in humans is unknown, we performed a study to determine the infective dose of the parasite in healthy adults.After providing informed consent, 29 healthy volunteers without evidence of previous C. parvum infection, as determined by the absence of anti-cryptosporidium-specific antibodies, were given a single dose of 30 to 1 million C. parvum oocysts obtained from a calf. They were then monitored for oocyst excretion and clinical illness for eight weeks. Household contacts were monitored for secondary spread.Of the 16 subjects who received an intended dose of 300 or more oocysts, 14 (88 percent) became infected. After a dose of 30 oocysts, one of five subjects (20 percent) became infected, whereas at a dose of 1000 or more oocysts, seven of seven became infected. The median infective dose, calculated by linear regression, was 132 oocysts. Of the 18 subjects who excreted oocysts after the challenge dose, 11 had enteric symptoms and 7 (39 percent) had clinical cryptosporidiosis, consisting of diarrhea plus at least one other enteric symptom. All recovered, and there were no secondary cases of diarrhea among household contacts.In healthy adults with no serologic evidence of past infection with C. parvum, a low dose of C. parvum oocysts is sufficient to cause infection.Keywords:
Cryptosporidium parvum
Infectivity
Diarrheal diseases
Cryptosporidium parvum presents a significant problem for the water industry and public health officials because of its prevalence in sources of drinking water and its resistance to chlorine-based disinfectants; there is an urgent need for alternative, more effective disinfection strategies. Therefore, developing and evaluating methods for assessing the infectivity and inactivation of C. parvum oocysts are of paramount importance. Infectivity assays based on in-vitro cell culture have been developed as alternatives to human and animal-based assays to overcome ethical, cost, and practicality issues. Data obtained over a two-year period with an HCT-8 cell culture/RT-PCR infectivity assay generated an ID50 of 99 oocysts (95% CI: 84-117) and demonstrated that the cell culture assay was equivalent to the standard CD-1 mouse model for measuring infectivity of C. parvum oocysts. Aggregate data generated over two years using the HCT-8 cell culture/RT-PCR assay to measure UV disinfection of C. parvum demonstrated that 2.4 mJ/cm2 and 4.9 mJ/cm2 were necessary to achieve 1-log10 and 2-log10 inactivation, respectively. This work demonstrated that an HCT-8 cell culture-based infectivity coupled with RT-PCR for detecting C. parvum infections is a practical tool that can provide valuable information about the efficacy of disinfectants and the infectivity of oocysts in environmental waters.
Infectivity
Cryptosporidium parvum
Cite
Citations (0)
ABSTRACT. Cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD‐I mice using standardized protocols and a well‐characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose‐response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose‐response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.
Infectivity
Cryptosporidium parvum
Cite
Citations (43)
ABSTRACT In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium . For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.
Infectivity
Immunofluorescence
Cite
Citations (32)
The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.
Infectivity
Cryptosporidium parvum
Giardia
Ex vivo
Cite
Citations (64)
Cryptosporidium parvum is a well-known waterborne and opportunistic intracellular protozoan parasite that causes diarrheal illness. In this study, we quantitatively investigated reduction of the infectivity of C. parvum after gamma irradiation and repair of the infectivity during incubation time after irradiation. C. parvum oocysts were subjected to gamma irradiation at various doses (1, 5, 10, and 25 kGy), and the in vitro infectivity was measured by real-time PCR every day up to 7 days after irradiation. The in vitro infectivity of C. parvum on human ileocecal adenocarcinoma cells (HCT-8) was effectively reduced (> 2 log10) by irradiation at 10 kGy or more. However, in the experiment to find out repair of the infectivity, recovery was not noted until day 7 post-incubation. Key words: Cryptosporidium parvum, gamma irradiation, infectivity
Infectivity
Cryptosporidium parvum
Gamma Irradiation
Cite
Citations (11)
The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.
Infectivity
Cryptosporidium parvum
Cite
Citations (28)
ABSTRACT We reexamined the finding of Neumann et al. (10) that intact Cryptosporidium parvum oocysts obtained after in vitro excystation were infectious for neonatal CD-1 mice. We used both established excystation protocols and our own protocol that maximized excystation (2). Although intact oocysts isolated after any of three protocols were infectious for neonatal CD-1 mice, the infectivity of intact oocysts isolated with our optimized excystation protocol was significantly lower than the infectivity of intact oocysts isolated after established protocols or from fresh oocysts. Excystation should not be considered a valid measure of C. parvum viability, given that it is biologically implausible for oocysts to be nonviable and yet infectious.
Infectivity
Cryptosporidium parvum
Cite
Citations (19)
An in vivo SCID mouse infectivity assay was used to determine its capacity to detect the infectivity of low concentrations of Cryptosporidium parvum oocysts in water. This biological test can be applied to demonstrate oocysts infectivity in water samples derived from drinking water supply and/or environmental sources. Ein biologisches Testverfahren zum Nachweis infektiöser Cryptosporidium parvum Oozysten in niedrigen Konzentrationen im Wasser Ein In-vivo-SCID-Maus-Übertragbarkeitstest wurde auf seine Eignung hin untersucht, infektiöse Cryptosporidium parvum Oozysten in niedrigen Konzentrationen in Wasser nachzuweisen. Es zeigte sich, dass dieses biologische Testverfahren sowohl für Wasserproben aus dem Wasserversorgungssystem als auch für andere Umweltproben einsetzbar ist.
Cryptosporidium parvum
Infectivity
Cite
Citations (13)
ABSTRACT Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.
Infectivity
Cryptosporidium parvum
Disinfectant
Peracetic acid
Propidium iodide
Cite
Citations (49)
Infectivity
Cryptosporidium parvum
Ultraviolet light
Ultraviolet
Cite
Citations (1)