Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-07-30
2001-06-05
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06242178
ABSTRACT:
This invention was made by the Centers for Disease Control and Prevention, an agency of the United States Government.
TECHNICAL FIELD
The present invention relates in general to the field of microbiology. In particular, the invention relates to the species-specific detection of Candida, using DNA probes generated from the internal transcribed spacer 2 region of thirteen Candida species.
BACKGROUND OF THE INVENTION
Candidiasis is a fungal infection of mucosal membranes and other tissues. The infection is caused by the yeast-like organism Candida. Numerous species of Candida exist, including
C. albicans
. Rapid diagnosis of Candida infection has become important in recent years due to a substantial rise in the incidence of candidiasis. This increase in candidiasis is most likely caused by the rising incidence of AIDS, more intensive regimens of cancer therapy, complications of abdominal or cardio-thoracic surgery, organ transplantations, burns and trauma. While most candidiasis patients are infected with
C. albicans
, the number of non-
C. albicans
infections has been growing steadily and may reflect the increased use of azole drug prophylaxis and therapy since some non-
C. albicans
species are innately resistant to these drugs. Additional risk factors commonly associated with the onset of candidiasis include protracted, broad-spectrum antibiotic therapies, invasive devices, and prolonged hospital stays. Under these conditions, an antibiotic resistant replacement flora, including one or more Candida species, can proliferate in the gastrointestinal tract and invade from mucosal foci to deep tissues, especially when mucosal integrity has been disrupted as a result of chemotherapy or surgery. As the number of risk factors increases, the odds of developing candidiasis multiplies. (Jarvis, W. R. 1995. Epidemiology of nosocomial fungal infections, with emphasis on Candida species.
Clin. Inf. Dis.
20:1526-30; Wenzel, R. P. 1995. Nosocomial candidemia: risk factors and attributable mortality.
Clin. Inf. Dis.
20:1531-4; Wingard, J. R. 1995. Importance of Candida species other than
C. albicans
as pathogens in oncology patients.
Clin. Inf. Dis.
20:115-25; and Fridkin, S. K. et al., 1996. Epidemiology of nosocomial fungal infections.
Clin. Micro. Rev.
9:499-511).
Candida species such as
C. glabrata
and
C. krusei
are emerging as the causative agents of candidiasis possibly because these species are innately less susceptible to azole drugs. In addition, the ability of species such as
C. parapsilosis
to survive in the hospital environment increases the possibility of nosocomial transmission. Consequently, rapid identification to the species level is necessary for more timely, targeted, and effective antifungal therapy, and to facilitate hospital infection control measures.
Current methods available in the clinical laboratory to identify Candida species rely on morphology and assimilation tests. These tests require approximately three to five days to complete, sometimes requiring additional tests. (Warren, N. G. and K. C. Hazen, 1995. Candida, Cryptococcus, and other yeasts of medical importance, p. 723-737. In P. R. Murray, E. J. Barton, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.) Manual of Clinical Microbiology, 6th ed., American Society for Microbiology, Washington, D.C.)
U.S. Pat. No. 5,426,027, incorporated herein by reference, describes a method for detecting five species of Candida using the polymerase chain reaction (PCR) technique. The five species detected by the method are
C. albicans, C. glabrata, C. tropicalis, C. parapsilosis
, and
C. krusei.
U.S. Pat. No. 5,426,027 provides a rapid method for the isolation, release, purification, amplification and detection of DNA from the above-identified species of Candida from blood and other body fluids. The method uses universal fungal primers that amplify the multi-copy internal transcribed spacer 2 region (ITS2) of rDNA, located between the 5.8S and the 28S rDNA coding regions, to enhance the amount of Candida target DNA in samples. Once amplified, target DNA is hybridized to probes which are then used in a microtitration plate format for the identification of specific species of Candida. However, the method described in U.S. Pat. No. 5,426,027 is incapable of differentiating between Candida species other than those listed above.
Due to the increase in infections caused by other species of Candida, and the differences in therapeutic strategies for treating candidiasis caused by various Candida species, it is desirable to have a method for detecting and differentiating additional Candida species.
Therefore, there is a need for sensitive, rapid, species-specific methods for the detection of Candida. In addition, there is a need for universal fungal primers to amplify all fungal DNA followed by species-specific probes for Candida to be used in detection methods and as scientific research tools to investigate the Candida organism and to implement appropriate therapies and treatments for candidiasis.
SUMMARY OF THE INVENTION
Isolated nucleic acid molecules that selectively hybridize with nucleic acid molecules encoding the internal transcribed spacer 2 (ITS2) region of various species of Candida, or complementary sequences thereof, are described herein. The nucleic acid molecules are useful as probes to detect the presence and identity of a Candida species in a sample or specimen with high sensitivity and specificity. The nucleic acid molecules are also useful as laboratory research tools to study the organism and related diseases and to guide therapies and treatments for those diseases. In addition, methods are described for the detection of and differentiation between Candida species.
The Candida species detected by the probes and methods described herein include
C. guilliermondii, C. haemulonii, C. kefyr, C. lambica, C. lusitaniae, C. norvegensis, C. norvegica, C. rugosa, C. utilis, C. viswanathii, C. zeylanoides, C. dubliniensis
, and
C. pelliculosa.
The method provides a simple, rapid, and feasible means for identifying and distinguishing these Candida species in clinical laboratories.
Therefore, it is an object of the present invention to provide probes and sensitive methods for detecting and differentiating Candida organisms in clinical and laboratory settings.
It is a further object of the present invention to provide nucleic acid probes specific for various Candida species.
It is a further object of the present invention to provide species-specific nucleic acid probes that hybridize to the internal transcribed spacer 2 region of particular Candida species.
It is a further object of the present invention to provide simple, rapid and reliable methods for detecting, diagnosing, or monitoring the progress of therapy for diseases caused by Candida organisms.
It is a further object of the invention to provide methods for detecting Candida in a tissue specimen or biological fluid sample of an infected patient, including a blood sample, or in cultures of Candida from any source.
These and other objects, features, and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
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Elie, C.M. et al., “Rapid Identification of Candida Species Using Species-Specific DNA Probes”, Abstract of the General Meeting of the American Society for Microbiology, vol. 97, No. 0, 1997, p. 161.
Williams, D.W. et al., “Identification of Ca
Elie Cheryl M.
Lott Timothy J.
Morrison Christine J.
Reiss Errol
Centers for Disease Control and Prevention
Jones W. Gary
Jones & Askew LLP
Whisenant Ethan
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