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

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.

1 . A reagent for the classification of fungi, comprising: 
 a probe set complementary to nucleic acid sequences of at least one fungal drug susceptibility-type.    
     
     
         2 . The reagent of  claim 1 , further comprising one or more additional probe sets complementary to nucleic acid sequences of other fungi of other drug susceptibility-types.  
     
     
         3 . The reagent of  claim 1 , wherein probe sets comprise one or more of nucleic acid probes, locked nucleic acid probes, peptide nucleic acid probes, or other nucleic acid probe mimics or analogues.  
     
     
         4 . The reagent of  claim 1 , wherein a fungal drug-susceptibility type comprises a full spectrum of response to a compound, wherein the full spectrum of response comprises one or more of susceptible; susceptible/intermediate; intermediate; susceptible/susceptible, dose-dependant; susceptible, dose-dependant; susceptible, dose-dependant/resistent; susceptible-dose/delivery dependent; intermediately resistant; or resistant.  
     
     
         5 . The reagent of  claim 1 , wherein the fungal drug susceptibility-type comprises resistance to an anti-fungal compound, or combination of compounds.  
     
     
         6 . The reagent of  claim 5 , wherein the anti-fungal compound or combination of compounds comprises one or more of fluconazole, caspofungin, voriconazole, and amphotericin B.  
     
     
         7 . The reagent of  claim 1 , wherein at least one fungal drug susceptibility type comprises one or more of a fluconazole-sensitive drug susceptibility-type; a fluconazole-sensitive, dose dependant drug susceptibility-type; or a fluconazole-resistant drug susceptibility-type.  
     
     
         8 . The reagent of  claim 7 , wherein the fluconazole-sensitive drug susceptibility-type comprise one or more of  Candida albicans  and/or  Candida parapsilosis.    
     
     
         9 . The reagent of  claim 7 , wherein the fluconazole-sensitive, dose dependent drug susceptibility-type comprise  Candida tropicalis.    
     
     
         10 . The reagent of  claim 7 , wherein the fluconazole-resistant drug susceptibility-type comprise one or more of  Candida glabrata  or  Candida krusei.    
     
     
         11 . The reagent of  claim 1 , wherein the fungi comprise caspofungin-,voriconazole- or amphotericin B-sensitive  
     
     
         12 . The reagent of  claim 1 , wherein the nucleic acid sequences comprise one or more of ribosomal RNA (including, but not limited to 5.8S, 18S and 26S sequences), ribosomal DNA, (including, but not limited to 5.8S, 18S and 26S sequences) or complements thereof.  
     
     
         13 . The reagent of  claim 1 , wherein at least a portion of a probe of the probe set is at least about 86% identical to the nucleobase sequence or complement thereof selected from SEQ. ID NOS 1-24.  
     
     
         14 . The reagent of  claim 13 , wherein the probe sequences comprise 8-17 subunits in length.  
     
     
         15 . The reagent of  claim 1 , wherein the probe set comprises at least one detectable moiety.  
     
     
         16 . The reagent of  claim 15 , wherein the detectable moiety or moieties comprise one or more of a conjugate, a branched detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester or a luminescent compound.  
     
     
         17 . The reagent of  claim 15 , wherein at least one probe is self-reporting.  
     
     
         18 . The reagent of  claim 17 , wherein the self-reporting probe comprises a PNA Linear Beacon.  
     
     
         19 . The reagent of  claim 1 , wherein at least one probe of the probe set is unlabeled.  
     
     
         20 . The reagent of  claim 1 , wherein at least one probe of the probe set is bound to a support.  
     
     
         21 . The reagent of  claim 1 , wherein at least one probe of the probe set further comprises a spacer or a linker.  
     
     
         22 . The reagent of  claim 1 , wherein in situ hybridization is used to analyze a sample for the presence of fungi.  
     
     
         23 . The reagent of  claim 1 , wherein at least two probes are differently labeled and wherein the probes are adapted to distinguish two or more drug susceptibility-types.  
     
     
         24 . The reagent of  claim 15 , wherein coincidental fluorescence is used to detect a fungal susceptibility-type.  
     
     
         25 . The reagent of  claim 1 , wherein the probe set comprises a PNA probe for fluconazole-sensitive fungi and a PNA probe for fluconazole-resistant fungi.  
     
     
         26 . The reagent of  claim 25 , wherein the fluconazole-sensitive fungi comprise one or more of  C. albicans  or  C. parapsilosis  and wherein the fluconazole-resistant fungi comprise one or more of  C. krusei  or  C. glabrata    
     
     
         27 . The reagent of  claim 1 , wherein the probe set comprises one or more of a PNA probe for fluconazole-sensitive fungi, a PNA probe for fluconazole-sensitive/dose-dependant fungi or a PNA probe for fluconazole-resistant fungi.  
     
     
         28 . The reagent of  claim 27 , wherein the fluconazole-sensitive fungi comprise one or more of  C. albicans  or  C. parapsilosis , the fluconazole-sensitive/dose dependant fungi comprise one or more of  C. tropicalis  and the fluconazole-resistant fungi comprise one or more of  C. krusei  or  C. glabrata    
     
     
         29 . The reagent of  claim 1 , wherein three or more differently labeled probe sets are used to distinguish between antifungal drugs.  
     
     
         30 . A method for determining the susceptibility-type of fungi, comprising: 
 contacting a sample with one or more probe sets, wherein the probe set comprises a complementary sequence to a nucleic acid sequence of at least one fungal drug susceptibility-type, and    correlating hybridization of a probe to an established susceptibility-type.    
     
     
         31 . The method of  claim 30 , wherein the hybridization is indicative of presence, identity and/or amount of microorganisms in the sample.  
     
     
         32 . The method of  claim 30 , wherein the probe sets comprise an azole sensitivity probe set and an azole resistance probe set.  
     
     
         33 . A method for selecting antifungal therapy, comprising: 
 a) contacting a sample with a probe set, wherein the probe set comprises a complementary sequence to a nucleic acid sequence of at least one fungal drug susceptibility-type;    b) hybridizing the probe set to a nucleic acid sample; and    c) detecting hybridization; and    d) selecting antifungal therapy based on hybridization, if any.    
     
     
         34 . A method for classifying fungi by therapy comprising 
 a) contacting a sample with a probe set, wherein the probe set comprises a complementary sequence to a nucleic acid sequence of at least one fungal drug susceptibility-type;    b) hybridizing the probe set to a nucleic acid sample; and    c) detecting hybridization; and    d) classifying the fungi based on hybridization.    
     
     
         35 . A multiplex method to select antifungal therapy, comprising: 
 a) contacting a sample with at least two probe sets, wherein the probe sets comprises complementary sequences to a nucleic acid sequences of at least one fungal drug susceptibility-type;    b) hybridizing the probe set to a nucleic acid sample; and    c) detecting hybridization; and    d) selecting antifungal therapy based on hybridization, if any.    
     
     
         36 . The multiplex method of  claim 35 , wherein the probe sets comprise one or more of a PNA probe set targeting  C. albicans  and  C. parapsilosis , a PNA probe set targeting  C. tropicalis , and a PNA probe set targeting  C. glabrata  and  C. krusei , wherein the PNA probe sets are independently labeled and wherein detection of hybridization of one or more of the PNA probes targeting  C. albicans  and  C. parapsilosis  indicates selection of fluconazole, detection of hybridization of one or more of the PNA probes targeting  C. tropicalis  indicates selection of increased dose of fluconazole and wherein detection of hybridization of one or more PNA probes targeting  C. glabrata  and  C. krusei  indicates selection of caspofungin, voriconazole, or amphotericin B  
     
     
         37 . The method of  claim 33 , wherein the analysis is in situ.  
     
     
         38 . The method of  claim 33 , wherein the analysis comprises fluorescence in situ hybridization.  
     
     
         39 . The method of  claim 33 , wherein the probes or their complementary sequences have been synthesized or amplified in a reaction.  
     
     
         40 . The method of  claim 33 , wherein results are generated in less than 8 hours.  
     
     
         41 . The method of  claim 33 , wherein results are generated in less than 3 hours.  
     
     
         42 . The method of  claim 39 , wherein nucleic acid synthesis or nucleic acid amplification reactions are selected from the group consisting of: Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Rolling Circle Amplification (RCA) and Q beta replicase.  
     
     
         43 . The method of  claim 33 , wherein the method further comprises adding at least one blocking probe to reduce or eliminate hybridization of the probe to a non-target sequence.  
     
     
         44 . The method of  claim 33 , wherein the nucleic acid sample is immobilized to a surface.  
     
     
         45 . The method of  claim 33 , wherein at least one probe of at least one probe set is immobilized to a surface.  
     
     
         46 . The method of  claim 45 , wherein the at least one probe is a component of an array.  
     
     
         47 . The method of  claim 33 , wherein the reagent comprises one or more of the probe sets of claims  16 - 29 .  
     
     
         48 . The method of  claim 33 , wherein the sample is a biological sample.  
     
     
         49 . The method of  claim 48 , wherein the biological sample is blood, urine, secretion, sweat, sputum, stool, mucous, or cultures thereof.  
     
     
         50 . A kit for selecting antifungal therapy, comprising: a) probe set comprising a complementary sequence to a nucleic acid sequence of at least one fungal drug susceptibility-type and b) other reagents or compositions necessary to perform the assay.  
     
     
         51 . The kit of  claim 50 , wherein the kit is used in an in situ hybridization assay.  
     
     
         52 . The kit of  claim 50 , wherein the kit is used for a real-time PCR assay.  
     
     
         53 . The kit of  claim 50 , wherein the kit is used to examine clinical samples or cultures thereof.