Cryptosporidium Inactivation by Low-Pressure UV in a Water Disinfection Device.

Abstract

Using animal infectivity tests, the authors evaluated a water disinfection device, UV Waterworks [TM] (UVW), for its ability to inactivate cryptosporidium parvum oocysts. The UVW employs low-pressure, germicidal ultraviolet (UV) light, delivering a dose of approximately 120 millijoules per square centimeter (mJ/[cm.sup.2]) under ideal water conditions at a flow rate of 4 gallons per minute (gpm). Dechlorinated tap water containing live oocysts was passed through the UVW at 4 gpm. The oocysts were captured on a filter, separated from the filter, and concentrated into inocula--10 microliters ([micro]L) each, containing between [10.sup.3]and [10.sup.7] oocysts--which were administered orally into 60 neonatal mice. After one week, the mice were killed, and sections of their terminal ilea were analyzed microscopically for signs of Cryptosporidium infection. In spite of the high dose of oocysts, none of the mice showed signs of infection. A process control run with the UV lamp off resulted in 95 percent infection a t a dose of [10.sup.3] oocysts per inoculum. The calculated reduction in oocyst infectivity from passage through UV Waterworks was at least 5.4 orders of magnitude. The authors conclude that exposure to low-pressure UV at 120 mJ/[cm.sup.2] effectively disables Cryptosporidium.

Editor's note:

Through NEHA's long-standing relationship with NSF International, NEHA was granted permission by NSF International to share with the Journal's readership various papers that were presented January 12-15, 2000, at the "NSF International Small Drinking Water and Wastewater Systems International Symposium and Technology Expo" in Phoenix, Arizona. This paper, "Cryptosporidium Inactivation by Low-Pressure UV in a Water Disinfection Device," is one of them.

It is important to note that these papers were screened by an NSF International advisory committee prior to their presentation at the conference, but they have not been peerreviewed by NEHA's Journal program for technical accuracy.

Because these papers contain useful and interesting ideas and information that might be either delayed or lost if the papers were sent through the Journal's normal peer review process, NEHA has decided to publish them as presented, with only minor editorial modifications.

We hope you look forward to more of these papers in future issues of the Journal!

Introduction

Certain pathogenic organisms, such as Cryptosporidium parvum, evade removal, resist chlorine disinfection, or do both in some municipal drinking-water treatment systems. They thus become a threat to immuno-compromised individuals consuming tap water. Ultraviolet (UV) disinfection is among the methods being investigated by researchers for treatment of municipal tap water to deactivate Cryptosporidium. In this paper, the authors report new results showing that low-pressure UV can be highly effective at inactivating this organism.

Cryptosporidium Viability Versus Infectivity A study that uses human hosts is the most accurate method of assessing the effect of any disinfection method on the infectivity to humans of Cryptosporidium parvum oocysts (DuPont et al., 1995). In practice, since such studies entail severe financial, legal, and ethical burdens, animal surrogates are used. Even animal studies are often, however, prohibitively expensive and time-consuming. Thus, several other methods have been developed to gauge the degree to which various disinfection treatments inactivate Cryptosporidium oocysts.

Two methods have been in common use in the past few years: the DAPI/PI (4',6'-diamidino-2-phenylindole and propidium iodide) vital-dyes assay (Campbell et al., 1992) and excystation (Finch et al., 1993a). These methods can demonstrate the viability of oocysts, and nonviable oocysts can be considered noninfective. A viable oocyst is not, however, necessarily infective. As explained below, equating viability with infectivity has in the past led to the false conclusion that UV light is relatively ineffective against Cryptosporidium.

Cryptosporidium and UV Disinfection

Initial studies of the effect of UV light on Cryptosporidium oocysts reported in the literature used only the vital-dyes and excystation methods. It was concluded that UV doses on the order of 10,000 mJ/[cm.sup.2] were needed to achieve a 2- to 3-log inactivation (Campbell et al., 1995). Pulsed, medium-pressure UV was later shown to result in a 2- to 3-log inactivation at around 2,000 mJ/[cm.sup.2], but a conventional, low-pressure UV system delivering 180 mJ/[cm.sup.2] was shown not to reduce the viability of oocysts significantly (Clancy et al., 1998a).

In spite of the disappointing results on the reduction in viability, several researchers recently have tested the effect of low doses of medium-pressure UV on the infectivity of oocysts. The results were surprising, showing a 3.9-log inactivation at only 19 mJ/[cm.sup.2] (Bukhari et al., 1999) and a greater-than-4-log inactivation at 41 mJ/[cm.sup.2] (Clancy et al., 1998b). Unpublished results of collimated-beam experiments using low-pressure UV and animal infectivity in early 1999 indicated that greater-than-4-log inactivation could be achieved at only 16 mJ/[cm.sup.2]. (Clancy Environmental Consultants, 1999). It was due to these encouraging results that the mouse infectivity assays reported here were performed.

Description of UV Waterworks [TM] (UVW)

UV Waterworks (UVW), a product of WaterHealth International, Inc., is an unpressurized disinfection unit designed for flow rates of up to 4 gallons per minute (gpm) (15 liters per minute [L/min]). Disinfection is achieved with a low-pressure mercury germicidal lamp suspended above an open-channel flow of water (Gadgil et al., 1997; Gadgil et al., 1998). Water flows through the unit by gravity. The mean residence time of water in the unit is 12 seconds at 4 gpm. The unit uses 60 watts of power and produces a UV fluence (or "dose") in the water of approximately 120 mJ/[cm.sup.2] under ideal conditions (water with turbidity less than 1 nephelometric turbidity unit [(NTU)] and UV transmittance greater than 95 percent at 1 cm). The fluence under such conditions was determined through biodosimetry with Bacillus subtilis spores according to the procedure outlined in Annex B and Annex C of NSF Standard 55 (NSF, 1991).

Method

The purpose of this experiment was to measure the effect of UVW on Cryptosporidium parvum oocyst infectivity with neonatal mice as hosts. To isolate the effect of UV light from possible influences of the physical passage of oocysts through the UVW, a process control was run in which the UV lamp was turned off. A trip control, consisting of oocysts that were not exposed to any of the experimental procedures prior to preparation of the mouse inocula, was also run to ensure that oocyst infectivity was not being reduced significantly by any extraneous factor.

Test Setup

The experimental setup used to pass the oocysts through the UVW is shown in Figure 1. Tap water was filtered through 1.0-and 0.5-micron ([micro]m) thermally bonded polyolefin fiber filters (Corning CoaX [TM]), and any residual chlorine was allowed to degas to the atmosphere over a period of three days. The turbidity was measured to be less than 0.1 NTU. The transmittance of 254 nanometers (nm) UV was 95 percent over a path 1 cm long. The filtered, dechlorinated water went into the 60-liter feed tank. The bottom delivery port of the tank was connected to the UVW inlet via a centrifugal pump and a valved flow meter. Just upstream of the UVW inlet, a spike injection port equipped with a check valve was incorporated into the line feeding into the UVW. This port was connected to a graduated spike cylinder via a metering diaphragm pump.

The effluent from the UVW was directed into a 90-L capture tank. A self-priming centrifugal pump sucked water from the capture tank through a 1.0-[micro]m (absolute) polycarbonate track-etch membrane filter (Corning Nuclepore [TM]) held in a capture filter housing 293 millimeters (mm) in diameter and discharged the filtered water to the drain.

Oocyst Exposure to UV and Process Control

Cryptosporidium parvum oocysts of the Harley Moon Isolate (National Animal Disease Center, Ames, Iowa) were obtained fresh (less than one month old) from the University of Arizona. This particular isolate had been maintained by passage through neonatal calves. A pre-enumerated stock solution containing 1 x [10.sup.8] oocysts per mL was divided into two equal 10-mL portions. One portion was assigned to the UV exposure test. A 100-[micro]L aliquot was withdrawn from the second portion to serve as the trip control. The rest of the second portion was reserved for the process control.

The vial containing the pre-enumerated oocysts (one for the UV exposure, another for the process control) was vortexed for 30 seconds and emptied into the spike cylinder, shown in Figure 1. The vial was then rinsed with filtered, dechlorinated tap water and emptied into the cylinder three times. Filtered, dechlorinated water was added to the cylinder to a total volume of 100 mL and thoroughly mixed.

Ten minutes before the start of the UV exposure test, the UV disinfection unit was turned on so that the UV lamp would warm up to its normal operating temperature. For the process control, the unit was not turned on (electrical power was not needed for the water to pass through, only for the UV lamp to be on). The system feed pump then began pumping water into the UVW from the feed tank at 4 gpm (15 L/min). When the flow was established, the diaphragm pump began pumping the 100-mL spike dose of oocysts into the feed stream at 200 mL/min. After the spike cylinder was emptied, additional volumes of filtered water were added over the course of two minutes to rinse the cylinder and spike plumbing. The main flow of water to the UVW was continued for one minute beyond the termination of the spike system rinsing to ensure that close to all oocysts were flushed out of the UVW.

The treated (UV-exposed or process-control) oocysts and water were collected in the capture tank. The centrifugal pump drew the entire tank volume plus a deionized water rinse of the tank walls through the capture filter. The filter membrane, with the trapped oocysts on its surface, was removed from the filter housing and placed in a beaker containing 100 mL of buffered solution. After the membrane was washed in this solution for two minutes by hand, was sonicated for five minutes, and was again washed by hand for two minutes, it was removed and discarded.

The solution remaining in the beaker contained the treated oocysts. It was centrifuged at 1,050g and aspirated to a l-mL pellet. This pellet was chilled and shipped overnight to the infectivity laboratory. It was kept chilled up to the time of the initiation of the infectivity assay, which was within 24 hours of the UV exposure.

Mouse Study

A bright-field hemacytometer was used to determine the concentration of oocysts present in the three vials containing the oocysts from the UV exposure, the process control, and the trip control, respectively. These concentrations were used to calculate and prepare the dilutions necessary to achieve the desired dose of oocysts in each of the mice inocula. The inocula were prepared from serial dilutions of the pre-enumerated starting suspension with sterile water adjusted to pH 7. Calibrated pipettors were used for all dilutions, Six sub-sample counts of the diluted oocysts were made to verify the concentration fed to the mice. The counts were performed and cross-checked by two technicians.

The mice used for this study were four-to-six-day-old neonatal CD-1 outbred mice. Approximately 60 mice were used in each of the three study conditions (trip control, process control, UV exposure). Of these 60, approximately 20 received a low dose of oocysts, 20 received a medium dose, and 20 received a high dose. To make possible detection of a potential reduction in oocyst infectivity of up to six orders of magnitude, target doses of the UV exposed oocysts were [10.sup.3], [10.sup.5], and [10.sup.7] per inoculum. The target doses of the process-control oocysts were [10.sup.2], [10.sup.3], and [10.sup.4] to allow for a 1- to 2-log reduction in infectivity due to factors other than UV light. Trip-control oocysts were expected to show very little loss in infectivity. Thus, their target doses were chosen to span the useful range of the reference dose-response function, which relates the dose of infective oocysts to the fraction of mice that become infected (Figure 2).

The oocysts were administered orally in 10[micro]L doses with a dedicated, calibrated pipettor equipped with a standard tip. The mouse was held in the palm of the technician's hand until the entire dose had been ingested. After seven days the animals were killed, and approximately 2 cm of each terminal ileum was excised. The tissue samples were fixed in formalin, embedded in paraffin, sectioned, mounted on microscope slides, and stained. The stained samples were then examined for evidence of Cryptosporidium parasite life cycle stages in the borders of the intestinal villi. Specimens with parasites were scored positive, and those without parasites were scored negative.

Infectivity Calculations

The proportion of mice infected in each case was used in conjunction with the reference dose-response function to calculate the number of infective oocysts in the inoculum. The model, illustrated graphically in Figure 2, was determined previously from experiments dedicated to this purpose (Korich et al., 1999). This model is very close to that determined by Finch et al. (1993b) in separate experiments.

Formula Used to Derive y, the Standard Error, in Equation 4

Y = [{[[s.sup.2].sub.yx][l/n + [(x - [x.sub.mean]).sup.2]/[[sigma].sub.i] = l..n[([x.sub.i] - [x.sub.mean]).sup.2])}.sup.1/2],

where

x = log(infective dose),

[[s.sup.2].sub.yx] = l/(n -2) x [[sigma].sub.i] =

l..n[([y.sub.j,predicted] - [[y.sub.j,measured]).sup.2],

[x.sub.i] and [y.sub.j] = the experimental data points of Korich et al. (1999) that generated eqoatinn(3),

The proportion of mice infected (P) was first converted to the "response logit" (Y):

and [x.sub.mean] = the mean of the [x.sub.i].

response logit = Y = ln[P/(1 - P)J (1)

The Korich et al. (1999) model, illustrated in Figure 2, is as follows:

Y= -6.752 + 3.611 x log(infective dose) (2)

That model was then used to calculate the number of infective oocysts, as follows:

infective dose = 10(Y + 6.752)/3.611 (3)

As shown in Figure 2, a dose of approximately 75 infective oocysts results in 50 percent of mice becoming infected.

The 90 percent confidence limit (CL) for each experimental logit response value Y was calculated as follows:

[CL.sub.y] Y [+ or -] [t.sub.0.1],[n-2] [X.sub..y] (4)

where

t is the value of the Student's t-distribution at the 10 percent and 90 percent limits,

n = 23 is the number of observations used to arrive at thhe Korich et al. (1999) linea regression [n - 2] is the number of degrees of freedom), and

.Y is the standard error, based on the Korich regression (see sidebar at left), of the logit response Y.

The upper and lower confidence limits were then inserted as Y values into Equation 3 to calculate the 90 percent confidence limits on the infective dose.

The response logit function does not allow for 0 percent (P = 0) or 100 percent (P = 1) of mice to be infected. For the purposes of quantification in this analysis, when P = 0 was encountered, one mouse was assumed to have been infected. When P = 1 was encountered, the number of mice infected was assumed to be one less than the total number in that batch. The confidence limits were not calculated in these cases.

Results

The quantitative results are summarized in Table 1 and Table 2. The trip control showed a reduction in oocyst infectivity of between 0.3 and 0.6 logs. The reduction might be due to some aspect of the handling, transport, or storage of these microorganisms from the time they were collected to the time they were administered to the mice. The other possibility is that the apparent low rate of infectivity reflects a difference between the mice used in this study and the mice used to generate the Korich et al. (1999) model--that is, a manifestation of the natural variability in the susceptibility of mouse populations.

As expected, the process control showed a slightly higher loss of infectivity overall than the trip control. This result is probably due to the more numerous environmental changes that the process-control oocysts experienced during the experiment. Nevertheless, at an inoculum dose of 1,087 oocysts, 95 percent of mice were infected, and at an inoculum dose of 9,500 oocysts, 100 percent of mice were infected.

By contrast, the UV-exposed oocysts were not able to produce infection in any mice at any of the three doses ([10.sup.3], [10.sup.5], and [10.sup.7] oocysts per inoculum). If a single mouse had been infected at the highest oocyst dose, the reduction in oocyst infectivity would have been six orders of magnitude.

Conclusions

Cryptosporidium parvum oocysts that were passed through a UV water disinfection device delivering approximately 120 mJ/[cm.sup.2] were effectively disabled. The UV-treated oocysts did not produce infection in any of the 60 mice to which they were administered. From the number of oocysts in the inoculum, the authors observed a reduction of greater than six orders of magnitude in the number of infective oocysts. When the maximum log reduction resulting from the three trip-control trials was subtracted, the effective log reduction from passage through the UVW alone would be at least 5.4 logs.

These results demonstrate that low-pressure UV at fairly low doses can be as effective against Cryptosporidium oocysts as it is against most bacteria and viruses. Previous results suggesting the contrary were limited because they came from studies of viability that used only staining or excystation techniques. There is now ample evidence that lack of oocyst viability as measured by these techniques is not a good indicator of infectivity. More studies like this one are needed to assess the effectiveness of UV disinfection against other harmful cysts and protozoans.

Acknowledgements: The experimental work was performed by Clancy Environmental Consultants and the University of Arizona's Sterling Parasitology Laboratory.

(Adapted with permission from NSF Proceedings of the Small Drinking Water and Wastewater Systems International Symposium and Technology Expo, held January 12-15, 2000, Phoenix, Arizona.)

Corresponding Author: Dr. Ashok J. Gadgil, WaterHealth International, Inc., Research Laboratory, 1700 Soscol Ave., Suite #5, Napa, CA 94559.

(*) Editor's note: Because this paper was originally published in the NSF Proceedings of the Small Drinking Water and Wastewater Systems International Symposium and Technology Expo, the references do not conform to normal Journal of Environmental Health style.

REFERENCES

Bukhari et al., "Medium-Pressure UV for Oocyst Inactivation," J. AWWA, 91(3), pp. 86-94, 1999.

Campbell, A.T., et al., "Viability of Cyrptosporidium parvum Oocysts: Correlation of In Vitro Excystation with Inclusion or Exclusion of Fluorogenic Vital Dyes," Applied and Environmental Microbiology, 58(11), pp. 3488-3493, 1992.

Campbell, AT., et al., "Inactivation of Oocysts of Cryptosporidium parvum by Ultraviolet Irradiation," Water Research, 29, pp. 25832586, 1995.

Clancy Environmental Consultants, personal communication, March 22, 1999.

Clancy, J.L., et al., "UV Light Inactivation of Cryptosporidium Oocysts," Journal AWWA, 90(9), pp. 92-103, 1998a.

Clancy, J.L., etal., "Inactivation of Cryptosporidium parvum Oocysts by Medium-Pressure Ultraviolet Light," in Proceedings of Providing Safe Drinking Water in Small Systems: Technology, Operations and Economics, JA. Cotruvo, G.F. Craun, and N. Hearne, Washington, D.C., pp. 151-154, 1998b.

DuPont, H.L., et al,, "The Infectivity of Cryptosporidium parvum in Healthy Volunteers," New England Journal of Medicine, 332(13), pp. 855-859, 1995.

Finch et al., "Ozone Inactivation of Cryptosporidium parvum in Demand-Free Buffer Determined by In Vitro Excystation and Animal Infectivity," Applied and Environmental Microbiology, 59(12), pp. 4203-4210, 1993a.

Finch et al., "Dose Response of Cryptosporidium parvum in Outbred Neonatal CD-1 Mice," Applied and Environmental Microbiology, 59(11), pp. 3661-3665, 1993b.

Gadgil, A., Greene, D., Drescher, A., Miller, P, and Kibata, N., "Low-cost Ultraviolet Disinfection System for Developing Countries," in Proceedings of Providing Safe Drinking Water in Small Systems: Technology, Operations and Economics, J.A. Cotruvo, G.F. Craun, and N. Hearne, Washington, D.C., pp. 165-172, 1998.

Gadgil, A., Dreseher, A., Greene, D., Miller, P, Motau, C., and Stevens, F, "Field-testing UV Disinfection of Drinking Water," in Proceedings of the 23rd WEDC Conference, Water and Sanitation for All, Water Engineering and Development Center of University of Loughborough, UK, pp. 153-156, conference held in Durban, South Africa, September 1-5, 1997.

Korich, D. G., et al., "Inter-laboratory Comparison of the CD-1 Neonatal Mouse Dose Response Model for Cryptosporidium parvum Oocysts," accepted for publication in J. Eukaryotic Microbiology, 1999.

NSF, Ultraviolet Microbiological Water Treatment Systems, ANSI/NSF 55-1991, American National Standard/NSF International Standard, NSF International, Michigan, USA, 1991.

[Graph omitted]

TABLE 1

Summary of Experimental Results--Controls


Oocyst Treatment                   Trip Cntrol [a]

Oocyst dose [+ or -] (6 counts)    241 [+ or -] 68  139 [+ or -] 42
Mice inoculated                    21               21
Mice infected                      8                6
Proportion infected                0.38             0.29
Infective oncyst dose              54               41
Log reduction in infectivity       0.6              0.5
Log reduction in infectivity: 90%  0.5, 0.8         0.3, 0.7
CLs (min, max)




Oocyst Treatment

Oocyst dose [+ or -] (6 counts)    46 [+ or -] 24
Mice inoculated                    20
Mice infected                      3
Proportion infected                0.15
Infective oncyst dose              25
Log reduction in infectivity       0.3
Log reduction in infectivity: 90%  0.1, 0.5
CLs (min, max)




Oocyst Treatment                   Process Control [b]

Oocyst dose [+ or -] (6 counts)    9,500 [+ or -] 840
Mice inoculated                    23
Mice infected                      23
Proportion infected                [greater than]0.96 [c]
Infective oncyst dose              [greater than]562 [c]
Log reduction in infectivity       [less than]1.2
Log reduction in infectivity: 90%  NA
CLs (min, max)




Oocyst Treatment

Oocyst dose [+ or -] (6 counts)    1,087 [+ or -] 87  115 [+ or -] 17
Mice inoculated                    20                 19
Mice infected                      19                 1
Proportion infected                0.95               0.05
Infective oncyst dose              484                12
Log reduction in infectivity       0.4                1.0
Log reduction in infectivity: 90%  0.2, 0.5           0.8, 1.2
CLs (min, max)



NA = not applicable.

SD = standard deviation.

(a)Oocysts not subjected to experimental conditions.

(b)Oocysts subjected to all experimental except UV exposure.

(c)The logit model was not valid for a 100% infection rate; in this
case, the calculation was performed with the assumption that one mouse
was not infected.
TABLE 2

Summary of Experimental Results--UV Exposure


Oocyst Treatment         Exposed to UV at [sim]12
                         mJ/[cm.sup.2]

Oocyst dose [+ or -] SD  1.19 ([+ or -] 0.10 x
 (6 counts)              [10.sup.7]
Mice inoculated          16
Mice infected            0
Proportion infected      [less than]0.0625 [a]
Infective oocyst dose    [less than]13 [a]
Log reduction in         [greater than]6.0 [a]
 infectivity
Log in reduction in      NA
 infectivity: 90% CLs
 (min, max)




Oocyst Treatment


Oocyst dose [+ or -] SD  1.19 ([+ or -] 0.10 x  1,194 [+ or -] 202
 (6 counts)              [10.sup.5]
Mice inoculated          21                     23
Mice infected            0                      0
Proportion infected      [less than]0.0476 [a]  [less than]0.0434 [a]
Infective oocyst dose    [less than]15 [a]      [less than]14 [a]
Log reduction in         [greater than]4.0 [a]  [less than]2.1 [a]
 infectivity
Log in reduction in      NA                     NA
 infectivity: 90% CLs
 (min, max)



(a)The logit response model was not valid for a 0% infection rate;
in this case, the calculation was performed with the assumption that
one mouse was infected.