LAB NEWS
October 1995 . . . . . . . . . . Vol. 38 No. 1

CRYPTOSPORIDIUM PARVUM, DRINKING WATER AND PATIENT TESTING

C. parvum is transmitted by the fecal-oral route, beginning with human ingestion of the infective oocysts. As the parasite goes through its life cycle within the infected host, more infective oocysts are passed into the environment. However, oocysts appear to be produced in two varieties. Thin-walled cysts probably break open within the gastrointestinal tract of the host, releasing free sporozoites which can, in turn, infect other enterocytes within the same host. This is an autoinfective process that would permit amplification of infection, with serious implications for immunocompromised patients. Amplification and dissemination of strongyloidiasis is also known to occur under such conditions. The autoinfective process guarantees that chronic persistent disease continues even in the absence of repeated exposure to more infective oocysts.

Thick-walled cysts are passed into the environment. They are infective and relatively resistant to both chlorine and ozone. Unfortunately infection is common among farm animals. When run off from farm lands is able to contaminate groundwater, environmental contamination is not easily controlled.

Water Sources
Dupont et al. (1) recently reported that the calculated ID50(50% infectious dose) for a clone of Cryptosporidium parvum in human volunteers was 132 oocysts. A total of 29 normal volunteers participated in this feeding study. Five of six who received 500 oocysts became ill; 2/3 who received 300; 3/8 who received 100; and 1/5 who received 30.

There was no detectable secondary spread among household contacts. When present in finished water, Cryptosporidium parvum is generally found at very low concentrations (<1/L). The MMWR (2) reported the analysis of a Cryptosporidium workshop held at the CDC in Atlanta in September 1994. This report (2) summarizes the views of attendees concerning epidemiology, future research, the role for laboratory testing of drinking water, and drinking water recommendations for the highly immunocompromised patient. It was difficult to estimate the percent of Cryptosporidiosis contributed from drinking water compared to other routes of transmission, such as human to human (e.g., sexual activity, fecal-oral) and food to human.

Cryptosporidiosis is not a reportable disease in many states and even where present, it is under-reported. Moreover, physicians may not always order the appropriate diagnostic test and such testing may not be included in routine "O&P" examinations. Virtually all surface water contains Cryptosporidium and current treatment processes cannot guarantee its removal. The MMWR report states that im-munocompromised individuals may want to consume water that is either boiled, obtained from a subterranean protected source, or instead use processed bottled water.

Cryptosporidium oocysts are present in 65 to 97% of surface drinking water sources. In finished tap water oocytes have been found in 27 to 54% of communities surveyed. A number of outbreaks have occurred in the United States and other countries. In the U.S. all outbreaks have occurred in tap water that has met all federal regulations. Furthermore, all the water sources associated with these outbreaks have been filtered. However, both the U.S. and most other countries only require infrequent testing of tap water, on the order of 1 coliform test per month per 1000 population. Therefore, it is not surprising that coliform tests may not be positive in an outbreak situation because the tests are performed so infrequently they would likely miss a spike of oocysts passing through the water distribution system (3).

Water Testing and Treatments
Results of Cryptosporidium testing of drinking water cannot be used to make public health decisions because the procedure has extremely poor sensitivity and specificity (4, 5). The procedure for analysis of Cryptosporidium in drinking water is very complex. It yields many false positives, and it has a sensitivity of less than 20%. Also, the procedure cannot distinguish infectious from noninfectious, or viable from non-viable, oocysts. If performed, virtually all water utilities could find oocysts in their source surface water and one-quarter to one-half would find them in their finished water. Cryptosporidium is not found in subterranean (i.e., deep aquifer) water which is not subject to surface water influence. In the U.S. approximately 50% of public tap drinking water comes from surface water; however, human exposure to surface water is increased since surface and subterranean sources are often mixed. Filtration and coagulation used in the municipal water industry may not remove oocysts, which are also resistant to chlorination.

Those at serious risk for Cryptosporidiosis (i.e., AIDS patients, and others who are profoundly immunocompromised) should take drinking water precautions. First, they should not consume tap water that has surface water as its source, even in part. Secondly, they can boil their tap water for one minute. Thirdly, they can consume bottled water which has absolute source protection (meets European directive 80/777), or is subject to other barrier protection. These include frequent testing for coliforms in the source and finished product and one or more of the following: distillation, reverse osmosis, and 1 micron absolute filtration. Ozonation is effective against Cryptosporidia but the exact conditions and standards are still being established. Bottlers that are members of the International Bottled Water Association have manufacturing and plant inspection rules that provide for a substantial level of safety. Information regarding a particular bottled water can be obtained using the 800 telephone number printed on each bottle or from the bottler's address. Point-of-use devices which are 1 micron absolute as certified by the NSF should provide effective protection, but these must be maintained frequently, since they may concentrate and provide a growth medium for bacteria.

Clinical Disease
For an infected patient the level of immuno-competence will determine whether an acute and self-limited infection moves further on to cause low-grade fever, watery diarrhea and abdominal discomfort on an acute or chronic basis. For immunocompromised individuals Cryptosporidiosis may cause respiratory disease, cholecystitis, cholangitis, and pancreatitis. It is not known whether the debilitating diarrhea of Cryptosporidiosis is caused by a toxin or some other mechanism of damage to the infected enterocyte's microvillus border. Whatever the mechanism is for diarrhea production, it is markedly enhanced by immunosuppression particularly that associated with AIDS (6).

Diagnosis of Cryptosporidiosis
If an intestinal biopsy has already been performed as part of a patient's clinical work-up C. parvum can be observed by light microscopy using routine hematoxylin and eosin staining or electron microscopy. For stool samples our parasitology laboratory employs a modification of the acid-fast stain which utilizes dimethyl sulfoxide to enhance carbol fuchsin stain penetration and identify C. parvum oocysts (DMSO Modified Acid Fast Stain Kit, Trend Scientific, St. Paul MN). Size is important for identification of acid-fast oocysts to distinguish Cryptosporidia from the somewhat larger Cyclospora species.

FIGURE 1

Any stool sent for Cryptosporidium exam will receive a DMSO stain and an ELISA test for Cryptosporidium antigen. The stains will be done as usual and the antigens will be batched twice a week. If the DMSO stain is negative and the ELISA for Cryptosporidium is positive the following comment will be used: Although Cryptosporidia were not observed on direct staining, this sample tested positive using the more sensitive ELISA technique. Consider Cryptospiridia results positive for this sample. (Also any stool requested for ova and parasite exam for any patient on the Atkins Service or from the Nathan Smith Clinic will automatically receive a DMSO acid-fast stain and an ELISA test.)

Figure 1 shows our diagnostic work-up for stool specimens obtained from patients in whom cryptosporidiosis is suspect. We recently decided to supplement acid-fasting staining with a commercially available enzyme-linked immunosorbent assay (ProSpect Cryptosporidium) Microtiter Assay; Alexon Inc., Sunnyvale, CA). This allows us to confirm positive specimens and detect additional positive samples not observed with the modified acid-fast stain. Test specificity is based upon detection of a Cryptosporidium-Specific Antigen (CSA) which is produced by these parasites during their replication in the GI tract. The anti-CSA antibody is bound to wells in the plastic microtiter plates. This antibody captures any antigen present in stool samples. When the test is positive, a second anti-CSA antibody which is labeled with horseradish peroxidase attaches to the antigen in a sandwich allowing for spectrophotometric detection. The sensitivity of the assay appears to be greater than 95% and specificity is even greater. Cross-reactivity with other parasites is not a problem. Since parasitic infections are often found as multiple infections this is an important feature of such tests.

References

1. Dupont HL, Chappell CL, Sterling CR. The infectivity of cryptosporidium parvum in healthy volunteers. New Engl J Med 1995;332:855.
2. CDC. Assessing the health threat associated with waterborne cryptosporidiosis: Report of a workshop. MMWR 44: No. RR-6. 1-8.
3. Hardalo CJ. Safe water for travelers. Travel Medicine Advisor Update 1995;5:4.
4. Edberg SC. Association of indicators with Crypto-sporidium. J American Water Works Assoc. 1994; 4:98.
5. Clancy JL, et al. Reproductibility of Cryptosporidium assays. J. American Water Works Assoc. 1994; 86:89-97.
6. Topazian M. Bia FJ. New parasites on the block: emerging intestinal protozoa. Gastroenterologist 1994; 2:147.

Frank J. Bia, M.D., M.P.H.; Stephen C. Edberg, Ph.D., ABMM