Reagents, methods and kits for classification of fungi and direction of anti-fungal therapy

Inactive Publication Date: 2007-04-12
ADVANDX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] Described herein are reagents, methods and kits to identify and categorize fungi according to their established susceptibilities to antifungal agents. Embodiments of the invention utilize probes targeted toward fungal species in such a way that optimal therapy can be selected without necessitating identification of the organisms at the species level. By short-cutting

Problems solved by technology

Fungal infections, particularly those caused by yeasts, are associated with high morbidity and mortality.
Antifungal drugs are approved for, and directed towards the treatment of particular Candida species, however; identification to a species level is not commonly performed rap

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

PNA Probe Sequence

[0116] Fluconazole Sensitive Probe Set

Probe A-FamFam-OO-AGAGAGCAGCATGCA-NH2(Seq. Id. No. 1)

[0117] Fluconazole Resistant Probe Set

Probe B-TamTam-OO-GCAAGGGGCGCAAA-NH2(Seq. Id. No. 2)Probe F-TamTam-OO-GCAGCGGTGCGCAA-NH2(Seq. Id. No. 6)

(Note: Conventional nomenclature used to illustrate the termini of the PNA probe; O=8-amino-3,6-dioxaoctanoic acid; Fam=5(6)-carboxyfluorescein, Tam=5(6)-carboxytetramethyrhodamine.)

Strains

[0118] Overnight cultures of reference strains were prepared representing various Candida species including C. albicans, C. glabrata, C. guillermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, as well as baker's yeast, S. cerevisiae, and Staphylococcus epidermidis, a frequent blood culture contaminant.

Preparation of Smears.

[0119] For each strain, smears were prepared on a 8-mm diameter well of a Teflon-coated microscope slide (AdvanDx, Woburn, Mass.) by mixing one drop of culture with one drop of phosphate-buffe...

example 2

[0123] Example 2 was performed exactly as Example 1, except that a second probe was added to the Fluconazole Sensitive probe set.

PNA Probe Sequence

[0124] Fluconazole Sensitive Probe Set

Probe A-FamFam-OO-AGAGAGCAGCATGCA-NH2(Seq. Id. No. 1)Probe I-FamFam-OO-GAGAGTAACATACAA-NH2(Seq. Id. No. 9)

[0125] Fluconazole Resistant Probe Set

Probe B-TamTam-OO-GCAAGGGGCGCAAA-NH2(Seq. Id. No.2)Probe F-TamTam-OO-GCAGCGGTGCGCAA-NH2(Seq. Id. No.6)

(Note: Conventional nomenclature used to illustrate the termini of the PNA probe; O=8-amino-3,6-dioxaoctanoic acid; Fam=5(6)-carboxyfluorescein; Tam=5(6)-carboxytetramethyrhodamine.)

[0126] Probe-I-Fam was added in the hybridization solution at 500 nM, along with the other probes as described in Example 1. Microscopic examination was conducted using a fluorescence microscope equipped with a FITC / Texas Red dual band filter set. Fluconazole Sensitive fungi were identified by green fluorescent buds and Fluconazole Resistant fungi were identified by red fl...

example 3

[0130] Example 3 was performed exactly as Example 1, but with a different probe set.

PNA Probe Sequence

[0131] Fluconazole Intermediate Probe Set

Probe N-TamTam-OO-AAGAAGTAACATACA-NH2(Seq. Id. No. 14)

(Note: Conventional nomenclature used to illustrate the termini of the PNA probe; O=8-amino-3,6-dioxaoctanoic acid; Tam=5(6)-carboxytetramethyrhodamine.)

[0132] Probe N-Tam was added in the hybridization solution at 250 nM. Microscopic examination was conducted using a fluorescence microscope equipped with a FITC / Texas Red dual band filter set. Fluconazole Intermediate fungi were identified by red fluorescent buds. Results are recorded in Table 4.

TABLE 4Species Id.ResultIncidence1C. albicansNegative54.5%C. glabrataNegative12.3%C. kruseiNegative1.5%C. parapsilosisNegative17.8%C. tropicalisPositive / Red (Fluconazole Intermediate)9.5%Other Candida spp.2Negative4.5%S. epidermidisNegativeN / A

1Average incidence of Candida species for the United States, Europe, Canada, and Latin America. M....

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Abstract

Provided herein are methods, kits and compositions to classify fungi. Methods are provided for classification of fungi according to established phenotypes, for example, antimicrobial susceptibility profiles. More specifically, the invention provides methods for the use of PNA probes in diagnostic applications, which will aid in the direction of appropriate therapy against fungi.

Description

RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Ser. No. 60 / 719,153, filed Sep. 20, 2005 and U.S. Provisional Application Ser. No. 60 / 789,656, filed Apr. 5, 2006, which are hereby incorporated by reference in their entirety.BACKGROUND [0002] Fungal infections, particularly those caused by yeasts, are associated with high morbidity and mortality. An increase in the prevalence of nosocomial fungal infections, especially bloodstream infections (BSI) attributed to Candida species, has contributed to an overall increase in the proportion of BSI caused by yeast (Wisplinghoff CID 2004). [0003] The susceptibility of the various Candida species to available antifungal compounds is constantly being reexamined and results are widely published. The susceptibility of an isolate to a drug is often described in minimum inhibitory concentration units (MIC) which are often defined in terms of the species as a whole, though susceptibilities can vary widely...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/04
CPCC12Q2600/158C12Q1/6895
Inventor STENDER, HENRIKFIANDACA, MARK
Owner ADVANDX
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