Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-10-28
2002-11-05
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007700, C435S007920, C436S536000, C436S541000
Reexamination Certificate
active
06475747
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of detection assays and more particularly to an improved method for detecting
Cryptosporidium parvum
oocysts.
BACKGROUND OF THE INVENTION
Parasitic infections of the gastrointestinal tract are prevalent around the world. Many gastrointestinal parasites are transmitted by the consumption of contaminated food or water. Although gastrointestinal parasitic infections in the general population cause abdominal disorders for only a short period of time, in the immunocompromised individual, a parasitic infection can be deadly.
Cryptosporidium parvum
(
C. parvum
) is a food or waterborne parasite that infects humans and animals causing severe intestinal distress. Since the 1970's,
C. parvum
has been receiving increased world wide attention. In the early 1980's following two outbreaks of
C. parvum
infections in the United Kingdom resulting in a total of 516 cases, the British government was compelled to devise a method for detecting
C. parvum
in water. (K. M. Shepherd et al., APPLD. AND ENVIRON. MICRO., Vol. 62, No. 4 pp. 1317-1322 (1996)). In the United States, waterborne outbreaks of
C. parvum
are being reported with increasing frequency. One of the latest outbreaks took place in Milwaukee, Wis. in April 1993 involving the infection of an estimated 400,000 people. (C. Drozd et al. APPLD. AND ENVIRON. MICRO., Vol. 62, No. 4 pp. 1227-1232 (1996)). Infection caused by
C. parvum
is particularly dangerous because it can cause prolonged diarrheal illness that may be potentially fatal for immunocompromised individuals.
C. parvum
is a parasite that infects its host by invading the intestinal and urogenital systems.
C. parvum
organisms may be transmitted in a variety of ways including via contaminated food or water, animal to animal contact, via farm animals such as sheep and calves, or alternatively by oocysts in feces. Human infections generally result from zoonotic spread, person-to-person contact, fecal-oral contact, oral-anal contact or waterborne transmission. Although, cryptosporidiosis occurs worldwide, children, travelers to foreign countries, male homosexuals, and medical personnel caring for patients with the disease, are at particular risk. In developed countries, 1 to 4% of children with gastroenteritis harbor
C. parvum
oocysts; and in developing countries, 4 to 11% of such children have cryptosporidiosis. Apart from humans, Cryptosporidium infections are widespread in several other vertebrates such as mammals, reptiles and fish: and accordingly, the frequency of cryptosporidiosis is reported to be relatively high for animal handlers and veterinarian personnel.
Unlike other coccidia,
C. parvum
is found on the brush border of intestinal epithelium and not within deep intracellular regions. Typically,
C. parvum
organisms are small (2 to 6 &mgr;m) spherules that inhabit the microvillus border of the intestinal epithelium arranged in rows along the brush border of the jejunum. After introduction into the intestine,
C. parvum
sporozoites attach to the microvilli surfaces and reproduce by schizogony (asexually). The resulting infective oocysts are passed into the intestinal lumen and passed in the feces. Following ingestion of the oocysts by another vertebrate, the oocysts release sporozoites that attach themselves to the epithelial surface and initiate a new cycle of infection.
As
C. parvum
organisms invade the surface of intestinal cells, the host experiences symptoms such as reduced appetite, severe diarrhea and chronic fluid loss. In normal hosts, the onset of the disease is explosive, with profuse, watery diarrhea and abdominal cramping that lasts from 4 to 14 days following exposure. The symptoms generally persist for 5 to 11 days, and then rapidly abate as remission of the parasite occurs in about 10-15 days. However, in immunocompromised individuals, (i.e. marasmic and malnourished children, individuals with congenital hypogammaglobulinemia, those receiving immunosuppressants for cancer therapy or organ transplantation, and patients with AIDS), onset of the disease is more gradual and diarrhea is more severe, with daily fluid losses of up to 15 to 20 liters. Unless the underlying immunologic defect is corrected, the diarrhea may continue persistently or remittently for life. (Merck Manual, Chapter 15 p. 237 16th ed. (1992)).
There is no effective, specific anti-
C. parvum
therapy available at present. Although some patients have responded positively to therapy with conventional antibiotics such as spiramycin and paromomycin, the result of infection is frequently fatal for immunocompromised individuals. In fact, cryptosporidiosis is one of the predominant causes of death in immunocompromised patients.
In light of the potential disastrous consequences of
C. parvum
infection, sensitive, efficient methods for detecting
C. parvum
contamination are necessary. In humans, the typical source of cryptosporidiosis is contaminated water, therefore safeguarding water supplies is a primary goal. The United States Environmental Protection Agency has recognized the necessity for improved detection methods by initiating the establishment of mandatory guidelines for
C. parvum
levels in drinking water.
Currently available detection systems indicate that
C. parvum
organisms are observed in “spikes”; meaning that levels of
C. parvum
in samples collected upstream and downstream, from the same source of the contamination, may not be identical when simultaneous readings are made. Consequently,
C. parvum
levels recorded from one location may differ significantly from readings taken from the same location minutes later. Detection of
C. parvum
in water is further complicated because the initial source of infection is difficult to identify. An abnormally high
C. parvum
concentration may be caused by water run-off from contaminated farm or pasture land, or an infant's soiled diaper carelessly discarded into a stream.
Ideally, continuous filtration systems having the capability to capture and retain
C. parvum
organisms for subsequent analysis would be installed in all water supply reservoirs to allow for continuous monitoring. Unfortunately, filtration systems currently in use often have filtration cartridges that either fail to retain organisms, frequently become clogged with mud or sediment, or must be replaced or cleaned with a frequency that renders the cartridges impractical.
C. parvum
detection assays presently in use are cumbersome and frequently inaccurate. For example, most assay test samples begin as crude mixtures of
C. parvum
oocysts separated out from mud deposits collected by filters. The oocysts are isolated by processes involving centrifugation and ultrafiltration. Separating oocysts in this manner is often tedious and inefficient since each time the test sample is spun and filtered, oocysts are lost in the process, inevitably resulting in lack of sensitivity and related inaccuracies. Another significant disadvantage of such assays is the large amount of time required for processing test samples. For example, in order to improve the optical properties of test samples for detection, oocysts must be stained. Typically, staining and subsequent detection procedures can take up to four days. Furthermore, samples can be tested only in small increments (i.e. 50 &mgr;l), and the sensitivity of most currently available assays is very low. Generally at least 50,000
C. parvum
oocysts per milliliter must be present for a positive detection result. Therefore,
C. parvum
assays currently in use are generally inefficient, inaccurate and inconsistent.
Another barrier to effective Cryptosporidium screening concerns sample turbidity. The term “turbidity” refers specifically to the clarity or transparency of water and the effect that any suspended particles in the water may have on this clarity. Turbidity is determined by quantifying the amount of light allowed to pass through a sample and is measured in NTUs (nephelometric turbidity units). Many source water sites of public water
Arrowood Michael J.
Call Jeffrey L.
Johnson Patrick W.
Lee Yeuk-Mui
Tsang Victor C. W.
Klarquist & Sparkman, LLP
The United States of America as represented by the Department of
Turner Sharon L
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