Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-03-20
2000-04-25
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 29, 435 912, 435 34, C12Q 168
Patent
active
060542756
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for detecting microorganisms of the genus Cryptosporidium and more particularly Cryptosporidium parvum.
The protozoan parasite Cryptosporidium parvum is recognised as an important cause of diarrhoeal illness primarily in infants and young children, (although immunologically healthy adults are susceptible) and is associated with persistent diarrhoea and severe illness in malnourished children. It is also a serious opportunistic pathogen in immunocompromised individuals, causing severe and unremitting diarrhoea that is often intractable to therapy. Chronic cryptosporidiosis is reported in as many as 10% of persons with AIDS in the United States and there are currently no effective therapeutic strategies for treating Cryptosporidium infection.
Waterborne transmission of this enteric parasite is a major concern. The infective stage (oocyst) of Cryptosporidium is transmitted by the faecal-oral route, with infected individuals excreting Cryptosporidium oocysts. Animals as well as humans may serve as sources of environmental contamination and human infection. The oocyst is environmentally stable and is able to survive and penetrate routine wastewater treatment and is resistant to inactivation by drinking water disinfectants. There are several species of Cryptosporidium but Cryptosporidium parvum is believed to cause the majority of mammalian infections. Cryptosporidium parvum oocysts are resistant to chlorination procedures normally used for water treatment, and contamination of water supplies can cause massive outbreaks of the disease such as the 1994 outbreak of cryptosporidiosis in Milwaukee resulting in diarrhoeal illness in an estimated 403,000 people.
In the absence of effective drugs to treat this ubiquitous infection, the control and clinical management of cryptosporidiosis depends upon rapid, accurate and sensitive diagnosis of the presence of the parasite, both in clinical specimens and environmental samples.
Clinical diagnosis of Cryptosporidium is time consuming, insensitive and generally requires the skills of highly trained operators. It has recently been reported that the detection limits of conventional diagnostic techniques for Cryptosporidium were as low as 50,000 oocysts per gram of faeces and that mean oocyst losses ranged from 51.2% to 99.6%. Further, the most commonly used coprodiagnostic techniques may fail to detect cryptosporidiosis in many immunocompromised and immunocompetent individuals. Immunological-based detection methods using immunofluorescence assays, enzyme-linked-immunosorbent and immunofluorescent-based diagnostic tests have been developed, several of which are now commercially available. Enzyme-linked immunoassays, although quick and easy to perform, generally show low sensitivity ranging from 3.times.10.sup.5 to 1.times.10.sup.3 oocysts per gram of faeces and monoclonal antibodies have the ability to bind to other microorganisms, i.e., to stain nonspecifically. In addition, Cryptosporidium isolates have been shown to exhibit a great deal of antigenic variability and therefore diagnostic antibodies may not recognise all isolates.
Environmental detection of Cryptosporidium generally involves filtering large volumes of water and examining it microscopically for Cryptosporidium oocysts by various staining or immunolabelling techniques. However, the efficiency of oocyst recovery may be as low as 1.3 to 5.5%. Recently, an alternative means of harvesting oocysts by calcium carbonate flocculation has been described with improved recovery ranging from 68% to >80%. Specialised flow cytometry and cell sorting techniques have also been developed to detect oocysts in water samples with greater sensitivity than conventional fluorescence microscopy. Although these methods are significantly more sensitive and considerably faster than conventional methods, they are costly and still require the skills of highly trained technical operators.
The development of the polymerase chain reaction (PCR) has permitted specific and sensitive detection of patho
REFERENCES:
patent: 5770368 (1998-06-01), De Leon et al.
Morgan Una
Thompson Richard Christopher Andrew
Horlick Kenneth R.
Murdoch University
Taylor Janell E.
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