Nucleic acids for detecting Aspergillus species and other...

Details Nucleic acids for detecting Aspergillus species and other... Nucleic acids for detecting Aspergillus species and other...

2000-06-27

2002-04-16

Arthur, Lisa B. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C536S023100, C536S024320, C536S024300, C536S023700

Reexamination Certificate

active

06372430

ABSTRACT:

TECHNICAL FIELD
This application relates in general to the field of diagnostic microbiology. In particular, the invention relates to the species-specific detection of Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella,
Pseudallescheria boydii
(
Scedosporium apiospermum
), and Sporothrix species.
BACKGROUND OF THE INVENTION
In recent years, chemotherapy for hematological malignancies, and high-dose corticosteroid treatment for organ transplant recipients, along with the spread of AIDS, have greatly increased the number of immunocompromised patients (1, 12, 14, 43). Saprophytic filamentous fungi, such as Aspergillus, Rhizopus, and Mucor species, found in the environment and considered to be of low virulence, are now responsible for an increasing number of infections in the immunocompromised host (17, 20, 43). In addition, these infections are often fulminant and rapidly fatal in immunocompromised patients (7, 11, 12, 20, 44). Morbidity and mortality is extremely high; for example, aspergillosis has a mortality rate of approximately 90% (8, 11).
To complicate matters, diagnosis is difficult and symptoms are often non-specific (18, 27, 29, 42, 44). Antibody-based tests can be unreliable due to the depressed or variable immune responses of immunocompromised patents (2, 9, 18, 46). Antigen detection tests developed to date have fallen short of the desired sensitivity (2, 9, 38). Radiographic evidence can be non-specific and inconclusive (5, 29, 36), although some progress in diagnosis has been made with the advent of computerized tomography (40). However, definitive diagnosis still requires either a positive blood or tissue culture or histopathological confirmation (3, 21). An added complication is that the invasive procedures necessary to obtain biopsy materials are often not recommended in thrombocytopenic patient populations (37, 41).
Even when cultures of blood, lung or rhinocerebral tissues are positive, morphological and biochemical identification of filamentous fungi can require several days for adequate growth and sporulation to occur, delaying targeted drug therapy. Some atypical isolates may never sporulate, making identification even more difficult (23). When histopathology is performed on tissue biopsy sections, the morphological similarities of the various filamentous fungi in tissue make differentiation difficult (16). Fluorescent antibody staining of histopathological tissue sections is not specific unless cross-reactive epitopes are absorbed out which can make the resultant antibody reactions weak (14, 19). Therapeutic choices vary (7, 41, 44) making a test to rapidly and specifically identify filamentous fungi urgently needed for the implementation of appropriately targeted therapy. Early and accurate diagnosis and treatment can decrease morbidity and increase the chances for patient survival (6, 27, 39). Furthermore, identification of filamentous fungi to at least the species level would be epidemiologically useful (24, 31, 43, 47).
PCR-based methods of detection, which show promise as rapid, sensitive means to diagnose infections, have been used in the identification of DNA from Candida species (13, 15, 30) and some other fungi, particularly Aspergillus species (31, 33, 45). However, most of these tests are only genus-specific (28, 38) or are directed to detect only single-copy genes (4, 35). Others have designed probes to detect multi-copy genes so as to increase test sensitivity (31, 33) but in doing so have lost test specificity because they have used highly conserved genes, which detect one or a few species but which are also plagued with cross-reactivities to human, fungal or even viral DNA (25, 31, 33).
Therefore, it is an object of the invention to provide improved materials and methods for detecting and differentiating Aspergillus and other filamentous fungal species in the clinical and laboratory settings.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acids for detecting Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Rhizomucor, Absidia, Cunninghamella, Pseudallescheria (Scedosporium), and Sporothrix species. Unique internal transcribed spacer 2 coding regions permit the development of probes specific for five different Aspergillus species,
A. flavus, A. fumigatus, A. niger, A. terreus
, and
A. nidulans
. The invention thereby provides methods for the species-specific detection and diagnosis of Aspergillus infection in a subject. In addition, species probes have been developed for three Fusarium, four Mucor, two Penicillium, five Rhizopus and one Rhizomucor species, as well as probes for
Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii
(
Scedosporium apiospermum
), and
Sporothrix schenckii
. Generic probes for Aspergillus, Fusarium, and Mucor species have also been developed.
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.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a simple, rapid, and useful method for differentiating filamentous fungal species from each other and from other medically important fungi. This invention enables a rapid, simple and useful method to isolate fungal DNA from host samples, and to apply the species- and genus-specific probes for the diagnosis of a disease. Ultimately, these probes can be used for in situ hybridization or in situ PCR diagnostics so that the morphology of host tissue, and microorganisms, remain intact.
The invention provides nucleic acids containing regions of specificity for five Aspergillus, three Fusarium, four Mucor, two Penicillium, five Rhizopus and one Rhizomucor species as well as probes for
Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii
(
Scedosporium apiospremum
), and
Sporothrix schenckii
. These nucleic acids are from the internal transcribed spacer 2 (“ITS2”) region of ribosomal deoxyribonucleic acid (rDNA) of the genome of the aforementioned filamentous fungi. The ITS2 region is located between the 5.8S rDNA region and the 28S rDNA region.
In particular, the invention provides nucleic acids from
Aspergillus flavus
(SEQ ID NO:1),
Aspergillus fumigatus
(SEQ ID NO:2),
Aspergillus niger
(SEQ ID NO:3),
Aspergillus terreus
(SEQ ID NO:4),
Aspergillus nidulans
(SEQ ID NO:5),
Fusarium solani
(SEQ ID NO:6),
Fusarium moniliforme
(SEQ ID NO:7),
Mucor rouxii
(SEQ ID NO:8),
Mucor racemosus
(SEQ ID NO:9),
Mucor plumbeus
(SEQ ID NO: 10),
Mucor indicus
(SEQ ID NO:11),
Mucor circinilloides f. circinelloides
(SEQ ID NO:12),
Rhizopus oryzae
(SEQ ID NO:13 and NO:14),
Rhizopus microsportis
(SEQ ID NO:15 and 16),
Rhizopus circinans
(SEQ ID NO:17 and 18).
Rhizopus stolonifer
(SEQ ID NO:19),
Rhizomucor pusillus
(SEQ ID NO:20),
Absidia corymbifera
(SEQ ID NO:21 and 22),
Cunninghamella elegans
(SEQ ID NO:23),
Pseudallescheria boydii
(teleomorph of
Scedosporium apiospermum
) (SEQ ID NO:24, 25, 26, and 27),
Penicillium notatum
(SEQ ID NO:28), and
Sporothrix schenkii
(SEQ ID NO:29). These sequences can be used to identify and distinguish the respective species of Aspergillus, Fusariunm, Mucor, Rhizopus, and Penicillium, and identify and distinguish these species from each other and from
Absidia corymbifera, Cunninghamella elegans, Pseudallescheria boydii
(
Scedosporium apiospermum
), and
Sporothrix schenkii.
Furthermore, the invention provides isolated nucleic acid probes derived from GenBank nucleic acid sequences (for
Penicillium marneffei
and
Fusarium oxysporum
only) or from the above nucleic acid sequences which may be used as species-specific identifiers of
Aspergillus flavus
(SEQ ID NO:30 and 31),
Aspergillus fumigatus
(SEQ ID NO:32),
Aspergillus niger
(SEQ ID NO:33),
Aspergillus terreus
(SEQ ID NO:34),
Aspergillus nidulans
(SEQ ID NO:35),
Mucor rouxii
(SEQ ID NO:36),
Mucor plumbeus
(SEQ ID NO:37),
Mucor indicus
(SEQ ID NO:38),
Mucor circinilloides f. circinelloides
(SEQ I

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