Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-06-06
2001-01-30
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06180339
ABSTRACT:
FIELD OF INVENTION
The inventions described and claimed herein relate to the design and composition of two nucleic acid probes capable of detecting many different fungal organisms in clinical, food, environmental and other samples. The inventions described and claimed herein also relate to the design and composition of probes capable of specifically detecting and identifying Acremonium sp.,
Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus,
Beauveria sp., Bipolaris sp., Blastoschizomyces sp.,
Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis,
Chrysosporium sp., Cladosporium sp.,
Coccidioides immitis, Cryptococcus neoformans
var gattii serotype B,
Cryptococcus neoformans
serotype A,
Cryptococcus laurentii, Cryptococcus terreus,
Curvularia sp., Fusarium sp.,
Filobasidium capsuligenum, Filobasidiella
(Cryptococcus)
neoformans
var bacillispora serotype C,
Filobasidiella
(Cryptococcus)
neoformans
var neoformans serotype D,
Filobasidium uniguttulatum,
Geotrichum sp.,
Histoplasma capsulatum,
Malbranchea sp., Mucor sp., Paecilomyces sp.,
Paracoccidioides brasiliensis,
Penicillium species,
Pneumocystis carinii, Pseudallescheria boydii,
Rhizopus sp.,
Sporothrix schenkii, Scopulariopsis brevicaulis
sp.,
Scopulariopsis brumpti, Saccharomyces cerevisiae,
and
Trichosporon beigelii
in clinical, food, environmental and other samples.
Fungi are eukaryotic microorganisms that are universally distributed. While in nature fungi play a major role in the decomposition of plant materials, they are also responsible for spoilage of food, beverage and pharmaceutical preparations. Out of an estimated 100,000 species of fungi described by mycologists, approximately 150 species are pathogenic to man and animals. The increasing incidence of AIDS and the development of newer treatments for hematologic malignancies and organ transplants has lead to an increase in the number of immunocompromised patients. These patients have a high risk of developing fungal infections, which if not rapidly diagnosed and treated are capable of causing death in a matter of days. The number of antifungal drugs is limited and their toxic side effects on the patient are much higher than that of comparable antibacterial therapy. A rapid diagnosis of fungal infection and start of treatment is critical in these patients. Books by Kwon-Chung and Bennett, along with Sarosi and Davies, provide an overview into the medical importance of fungi.
Fungal organisms are identified by morphology and nutritional characteristics. Fungi may take anywhere from two days to several weeks to grow in culture and often the same organism can take radically different forms depending on the growth conditions. This makes timely identification difficult even for the classically trained expert and impedes the treatment of patients where rapid identification of genus and species is of medical advantage.
The incidence and distribution of major pathogenic fungi varies by geographic location.
Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pseudallescheria boydii
and
Sporothrix schenkii
represent some of the leading causes of mycotic infections.
Aspergillus fumigatus
is among the top three causes of systemic fungal infection treated in hospitals. It usually affects patients with organ transplants, acute leukemias and burns and can be rapidly fatal if not diagnosed quickly. With over 150 species of Aspergillus present in the soil, air and water, accurate detection of
Aspergillus fumigatus
becomes extremely important.
Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis
and
Aspergillus ustus
represent a majority of Aspergillus species seen in clinical specimens and their presence can cause diagnostic difficulties.
Aspergillus flavus, Aspergillus fumigatus
and
Aspergillus niger
have been linked with disease in humans, with
Aspergillus fumigatus
being the predominant pathogen in North America. A few immunologic tests exist for
Aspergillus fumigatus
but these have limited sensitivity and specificity. There are also reports of development of polymerase chain reaction based tests for
Aspergillus fumigatus
based on the amplification of the Asp fl antigen gene and a ribosomal intergenic spacer (Spreadbury et. al.). The Spreadbury technique is based on the PCR amplification of a 401 bp fragment spanning the large subunit rRNA/intergenic spacer region. This relies on a pair of primers to specifically amplify DNA from
Aspergillus fumigatus only,
and is of no utility in identifying other fungi.
Blastomyces dermatitidis
is present in the soil, usually in bird droppings and animal feces. Infections often occur at construction sites and the ensuing lung infiltration and pneumonitis are usually fatal in immunocompromised patients. Diagnosis by culture may take weeks, and the organism is occasionally mistaken for other fungi. Existing immunological diagnostic tests are unreliable, and there is a need for rapid and reliable DNA based diagnostic tests. Similarly,
Histoplasma capsulatum
exists in the soil and is known to have infected at least 20% of the population of North America. Most infections start in the lung and resolve spontaneously, but may occasionally spread to other organs. AIDS patients represent a growing number of cases of Histoplasmosis. Diagnosis is difficult as immunological tests are often negative during the first 4-6 weeks of infection.
Coccidioides immitis
is found in abundance in the soil in Southwestern United States. Dust storms, farming, building construction, earthquakes and even hiking have been linked with outbreaks of disease. Lung infection followed by cavitation and disseminated miliary coccidioidomycosis are seen. Meningitis is usually lethal, and as with other fungi, mortality is highest in debilitated hosts. Four serotypes of
Cryptococcus neoformans
cause disease in humans. These are
Cryptococcus neoformans
serotype A,
Cryptococcus neoformans
var gatti serotype B,
Filobasidiella
(Cryptococcus)
neoformans
var bacillispora serotype C and
Filobasidiella
(Cryptococcus)
neoformans
var. neoformans serotype D. The incidence of this disease is growing rapidly, with up to 10% of HIV infected people developing cyptococcosis. DNA probes capable of detecting all 4 serotypes are required for the early diagnosis and treatment for life threatening infections like cryptococcal meningitis. A report by Stockman et. al. discusses commercial tests for Histoplasma, Blastomyces, Coccidioides, and Cryptococcus based on the 18S rRNA (Gen-Probe, Inc., San Diego, Calif.). The authors report sensitivities ranging from 87.8 to 100% and a specificity of 100%. One drawback of these probes is that these are used on rRNA extracted from fungal cultures. As some fungi may require up to 3 weeks to grow in culture, this technique cannot be used to expedite diagnosis until a culture becomes available.
Candida albicans
is one of the most common causes of fungal infection in humans. It is present in the respiratory, gastrointestinal and female genital tract of healthy individuals, and acts as an opportunistic pathogen in debilitated individuals on steroid or chemotherapy.
Diabetes mellitus
and indwelling catheters are other predisposing causes. Immunocompromised hosts show rapid hematogenous spread of fungi. Morbidity and mortality in untreated cases is high.
Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis
and
Candida tropicalis
are also known to cause disease in humans. DNA probes capable of identifying th
Kline Bruce C.
Sandhu Gurpreet S.
Bayer Corporation
Campbell Eggerton A.
Weingarten, Schurgin Gagnebin & Hayes LLP
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