Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• MIC minimum inhibitory concentration units
• species are described as resistant to a particular drug when a significant proportion of isolates tested have MIC values greater than the breakpoint, or when the recommended dosage of a drug is found to result in failure of treatment in a significant number of cases.
• Organisms which demonstrate intermediate susceptibilities are described as “susceptible, dose-dependant”.
• Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata , and Candida krusei account for greater than 95% of yeast isolates from blood (Pfaller JCM, Vol. 39, No.9. p. 3254-3259) and greater than 97% of nosocomial fungal infections (Wisplinghoff CID 2004).
• a trend in the relative proportions of these high prevalence Candida species has shifted towards a higher incidence of the “drug resistant” species C. glabrata and C. krusei in some patient populations (Trick et al CID 1998). It has been suggested that this change in species proportion reflects a selection of resistant nosocomial strains through the over use of empiric antibiotics.
• C. glabrata and C. krusei infections are generally treated with caspofungin, voriconazole, amphotericin B, or other strong antifungal compounds.
• 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 the traditional approach where optimal selection of therapy is awaiting species identification, information for selection of therapy is available faster without compromising the quality of the information.
• the invention circumvents the need for separating fungi which require the same treatment hereby simplifying both the analysis and the result interpretation.
• the methods and reagents provide information for the direction of therapy for treatment of fungal infections in a rapid and simple manner.
• the invention provides reagents and methods for selection of therapy against clinically prevalent Candida species.
• the invention provides PNA probes and probe sets targeted toward fungal species such that appropriate therapy can be selected without necessitating identification of the organisms to the species level.
• the test can provide therapeutic guidance for at least about 70% of yeast species associated with fungemia.
• a single 2-color, multiplex test can provide therapeutic guidance for at least about 70-85% of yeast species associated with fungemia.
• a single 3-color, multiplex test provides therapeutic guidance for at least about 95% of yeast species associated with fungemia.
• the reagents may be used in an array format rather than a multicolor format where the position of the signal, rather than the color of the signal, provides therapeutic guidance.
• the therapeutic guidance is for selection of a certain antifungal drug whereas in other instances the guidance is for avoiding certain antifungal drugs. Also, the guidance may be related to the dosing of a certain drug.
• Embodiments of the invention can provide information which is particularly useful when choosing between fluconazole and other anti-fungal drugs.
• absence of a signal by a particular test method provides useful diagnostic information, given that appropriate controls demonstrated that the method was capable of producing a positive result.
• reagents for the classification of fungi comprising a probe set complementary to nucleic acid sequences of at least one fungal drug susceptibility-type.
• the reagent further comprises one or more additional probe sets complementary to nucleic acid sequences of other fungi of other drug susceptibility-types.
• the 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.
• 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.
• the fungal drug susceptibility-type comprises resistance to an anti-fungal compound, or combination of compounds.
• the anti-fungal compound or combination of compounds comprises one or more of fluconazole, caspofungin, voriconazole, and amphotericin B.
• 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.
• the fluconazole-sensitive drug susceptibility-type comprises one or more of Candida albicans and/or Candida parapsilosis.
• the fluconazole-sensitive, dose dependant drug susceptibility-type comprises Candida tropicalis.
• the fluconazole-resistant drug susceptibility-type comprises one or more of Candida glabrata or Candida krusei.
• the fungi comprise caspofungin-, voriconazole- or amphotericin B-sensitive.
• 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.
• 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.
• the probe sequences comprise 8-17 subunits in length.
• the probe set comprises at least one detectable moiety.
• 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.
• At least one probe is self-reporting.
• the self-reporting probe comprises a PNA Linear Beacon.
• At least one probe of the probe set is unlabeled.
• At least one probe of the probe set is bound to a support.
• At least one probe of the probe set further comprises a spacer or a linker.
• in situ hybridization is used to analyze a sample for the presence of fungi.
• At least two probes are differently labeled and wherein the probes are adapted to distinguish two or more drug susceptibility-types.
• coincidental fluorescence is used to detect a fungal susceptibility-type.
• the probe set comprises a PNA probe for fluconazole-sensitive fungi and a PNA probe for fluconazole-resistant fungi.
• 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
• 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.
• 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
• the fluconazole-resistant fungi comprise one or more of C. krusei or C. glabrata
• three or more differently labeled probe sets are used to distinguish between antifungal drugs.
• 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.
• the hybridization is indicative of presence, identity and/or amount of microorganisms in the sample.
• the probe sets comprise an azole sensitivity probe set and an azole resistance probe set.
• methods 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.
• methods 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.
• methods 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.
• 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.
• the analysis is in situ.
• the analysis comprises fluorescence in situ hybridization.
• the probes or their complementary sequences have been synthesized or amplified in a reaction.
• results are generated in less than 8 hours.
• results are generated in less than 3 hours.
• 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.
• PCR Polymerase Chain Reaction
• LCR Ligase Chain Reaction
• SDA Strand Displacement Amplification
• TMA Transcription-Mediated Amplification
• RCA Rolling Circle Amplification
• Q beta replicase Q beta replicase
• the method further comprises adding at least one blocking probe to reduce or eliminate hybridization of the probe to a non-target sequence.
• the nucleic acid sample is immobilized to a surface.
• At least one probe of at least one probe set is immobilized to a surface.
• the at least one probe is a component of an array.
• the probe set comprises one or more of the PNA probe sets described herein.
• the sample is a biological sample.
• the biological sample is blood, urine, secretion, sweat, sputum, stool, mucous, or cultures thereof.
• kits 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.
• the kit is used in an in situ hybridization assay. In another embodiment, the kit is used for a real-time PCR assay.
• the kit is used to examine clinical samples or cultures thereof.
• nucleic acid targets which confer azole resistance to fungi.
• a multitude of molecular diagnostic methods are available for detection and identification of organisms involved in infectious disease. Amplification techniques, particularly those using the polymerase chain reaction (PCR) have been described for species identification for a wide range of bacteria and fungi. Amplification methods, probes and primers for detection of nucleic acid targets indicative of the presence of various fungi are described in several places (U.S. Pat. No. 6,858,387, US2003186259, U.S. Pat. No. 6,235,890). In addition, amplification methods have been described for detection of genes involved in antifungal resistance, see US2004185478.
• PNA Peptide nucleic acid probes
• FISH fluorescence in situ hybridization assays
• PNA probes have been described elsewhere for detection of Candida species (see US2003175727 incorporated herein by reference), however, as with all other methods for species identification, the time and complexity required for identification of Candida yeast to species often prevents immediate selection of the most appropriate therapy.
• ChromAgar is a novel, simple method which enables species identification of most clinically relevant species in a single test; however, it can take several days to get a result.
• the tools described herein could also be used to select safe, narrow spectrum, and high potency antifungal drugs that are often not selected due to the time constraints imposed by current speciation methods and susceptibility testing methods.
• drugs that may be indicated in this context are fluconazole, caspofungin, voriconazole, amphotericin B, or other anti-fungal drug or drugs.
• fluconazole caspofungin, voriconazole, amphotericin B, or other anti-fungal drug or drugs.
• an infectious organism which is resistant to the normal therapeutic dose of fluconazole belongs in the “resistant” susceptibility-type for fluconazole.
• a report of “susceptible” indicates that the pathogen is likely to be inhibited if the antimicrobial compound in blood reaches the concentrations usually achievable.
• a report of “intermediate” indicates that the result should be considered equivocal, and, if the microorganism is not fully susceptible to alternative, clinically feasible drugs, the test should be repeated.
• This category implies possible clinical applicability in body sites where the drug is physiologically concentrated or in situations where high dosage of drug can be used.
• This category also provides a buffer zone which prevents small uncontrolled technical factors from causing major discrepancies in interpretation.
• a report of “resistant” indicates that the pathogen is not likely to be inhibited if the antimicrobial compound in the blood reaches the concentrations usually achievable; other therapy should be selected.
• Drug susceptibility-type is assessed based on an expected result, which is derived from historical, empirical or experimental evidence.
• Preferred non-limiting methods for labeling PNAs are described in U.S. Pat. Nos. 6,110,676, 6,361,942, 6, 355,421, the examples section of this specification or are otherwise well known in the art of PNA synthesis and peptide synthesis.
• Non-limiting examples of detectable moieties (labels) suitable for labeling PNA probes used in the practice of this invention would include a dextran conjugate, a branched nucleic acid detection system, a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten, an acridinium ester and a chemiluminescent compound.
• Preferred haptens include 5 (6)-carboxyfluorescein, 2,4-dinitrophenyl, digoxigenin, and biotin.
• Preferred fluorochromes include 5 (6)-carboxyfluorescein (Flu), 6-((7-amino-4-methylcoumarin-3-acetyl) amino) hexanoic acid (Cou), 5 (and 6)-carboxy-X-rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2,3,3.5,5 and 5.5 are available as NHS esters from Amersham, Arlington Heights, Ill.), JOE, Tamara or the Alexa dye series (Molecular Probes, Eugene, Oreg.).
• Preferred enzymes include polymerases (e. g. Taq polymerase, Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNA polymerase 1 and phi29 polymerase), alkaline phosphatase (AP), horseradish peroxidase (HRP) and most preferably, soy bean peroxidase (SBP).
• polymerases e. g. Taq polymerase, Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNA polymerase 1 and phi29 polymerase
• AP alkaline phosphatase
• HR horseradish peroxidase
• SBP soy bean peroxidase
• probes that are used for the practice of this invention need not be labeled with a detectable moiety to be operable within the methods of this invention, for example when attached to a solid support
• Beacon probes are examples of self-indicating probes which include a donor moiety and a acceptor moiety.
• the donor and acceptor moieties operate such that the acceptor moieties accept energy transferred from the donor moieties or otherwise quench signal from the donor moiety.
• the acceptor moiety is a quencher moiety.
• the quencher moiety is a non-fluorescent aromatic or heteroaromatic moiety.
• the preferred quencher moiety is 4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl).
• the self-indicating Beacon probe is a PNA Linear Beacon as more fully described in U.S. Pat. No. 6,485, 901.
• the self-indicating probes of this invention are of the type described in WIPO patent application WO97/45539. These self-indicating probes differ as compared with Beacon probes primarily in that the reporter must interact with the nucleic acid to produce signal.
• spacers are used to minimize the adverse effects that bulky labeling reagents might have on hybridization properties of probes.
• Preferred spacer/linker moieties for the nucleobase polymers of this invention consist of one or more aminoalkyl carboxylic acids (e. g. aminocaproic acid), the side chain of an amino acid (e. g. the side chain of lysine or omithine), natural amino acids (e. g. glycine), aminooxyalkylacids (e. g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids (e. g. succinic acid), alkyloxy diacids (e. g. diglycolic acid) or alkyldiamines (e. g. 1, 8-diamino-3, 6-dioxaoctane).
• aminoalkyl carboxylic acids e. g. aminocaproic acid
• nucleic acid hybridization will recognize that factors commonly used to impose or control stringency of hybridization include formamide concentration (or other chemical denaturant reagent), salt concentration (i.e., ionic strength), hybridization temperature, detergent concentration, pH and the presence or absence of chaotropes.
• Optimal stringency for a probe/target sequence combination is often found by the well known technique of fixing several of the aforementioned stringency factors and then determining the effect of varying a single stringency factor. The same stringency factors can be modulated to thereby control the stringency of hybridization of a PNA to a nucleic acid, except that the hybridization of a PNA is fairly independent of ionic strength.
• Optimal stringency for an assay may be experimentally determined by examination of each stringency factor until the desired degree of discrimination is achieved.
• Blocking probes may also be used as a means to improve discrimination beyond the limits possible by optimization of stringency factors. Suitable hybridization conditions will thus comprise conditions under which the desired degree of discrimination is achieved such that an assay generates an accurate (within the tolerance desired for the assay) and reproducible result.
• Suitable in-situ hybridization or PCR conditions comprise conditions suitable for performing an in-situ hybridization or PCR procedure.
• suitable in-situ hybridization or PCR conditions will become apparent to those of skill in the art using the disclosure provided herein, with or without additional routine experimentation.
• Blocking probes are nucleic acid or non-nucleic acid probes that can be used to suppress the binding of the probing nucleobase sequence of the probing polymer to a non-target sequence.
• Preferred blocking probes are PNA probes (see: U.S. Pat. No. 6,110, 676). It is believed that blocking probes operate by hybridization to the non-target sequence to thereby form a more thermodynamically stable complex than is formed by hybridization between the probing nucleobase sequence and the non-target sequence. Formation of the more stable and preferred complex blocks formation of the less stable non-preferred complex between the probing nucleobase sequence and the non-target sequence.
• blocking probes can be used with the methods, kits and compositions of this invention to suppress the binding of the probes to a non-target sequence that might be present and interfere with the performance of the assay.
• Blocking probes are particularly advantageous for discrimination to the phylogenetically closest related species.
• Probe sets of this invention comprise one or more probes.
• one or more of the PNA probes of the set can be blocking probes.
• Probes sets may include any group of one or more of the probes of this invention, whether labeled or non-labeled, and may also include probes not specifically described here, but which include at least one of the probes of this invention.
• Preferred probe of the invention are listed in Table 1. TABLE 1 Sequence ID Name Nucleobase sequence Seq. Id. No. 1 Probe A AGA-GAG-CAG-CAT-GCA Seq. Id. No. 2 Probe B GCA-AGG-GGC-GCA-AA Seq. Id. No. 3 Probe C AGG-CAA-GGG-GCG-CA Seq. Id. No.
• Table 1 displays preferred probes of the invention. With reference to Table 1, the column on the left displays the sequence identification number, the center column displays the probe name, and the column on the right displays the nucleobase sequence of the probe.
• the PNA probes of this invention may comprise only a probing nucleobase sequence (as previously described herein) or may comprise additional moieties.
• additional moieties include detectable moieties (labels), linkers, spacers, natural or non-natural amino acids, or other subunits of PNA, DNA or RNA.
• Additional moieties may be functional or non-functional in an assay. Generally however, additional moieties will be selected to be functional within the design of the assay in which the PNA probe is to be used.
• the preferred PNA probes of this invention are labeled with one or more detectable moieties selected from the group consisting of fluorophores, enzymes and haptens.
• the probes of this invention are used in in situ hybridization (ISH) and fluorescence in situ hybridization (FISH) assays.
• ISH in situ hybridization
• FISH fluorescence in situ hybridization
• Excess probe used in an ISH or FISH assay typically must be removed so that the detectable moiety of the specifically bound probe can be detected above the background signal that results from still present but unhybridized probe.
• the excess probe is washed away after the sample has been incubated with probe for a period of time.
• self-indicating probes is a preferred embodiment of this invention, since there is no requirement that excess self-indicating probe be completely removed (washed away) from the sample since it generates little or no detectable background.
• self-indicating probes comprising the selected probing nucleobase sequence described herein are particularly useful in all kinds of homogeneous assays such as in real-time PCR or useful with self-indicating devices (e. g. lateral flow assay) or self-indicating arrays.
• Probe B-Tam Tam-OO-GCAAGGGGCGCAAA-NH 2 (Seq. Id. No. 2)
• Probe F-Tam Tam-OO-GCAGCGGTGCGCAA-NH 2 (Seq. Id. No. 6)
• 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-buffered saline containing 1% (v/v) Triton X-1 00 . The slide was then placed on a 55° C. slide warmer for 20 min at which point the smears were dry. Subsequently, the smears were disinfected by immersion into 96% (v/v) ethanol for 5-10 minutes and air-dried.
• FISH Fluorescence in situ hybridization
• Smears were covered with a drop of hybridization solution containing 10% (w/v) dextran sulfate, 10 mM NaCl, 30% (v/v) formamide, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) ficoll, 5 mM Na 2 EDTA, 1% (v/v) Triton X-100, 50 mM Tris/HCl pH 7.5 and 250 nM Probe A-Fam, 25 nM Probe B-Tam and 25 nM Probe F-Tam.
• Coverslips were placed on the smears to ensure even coverage with hybridization solution, and the slides were subsequently placed on a slide warmer (Slidemoat, Boekel, Germany) and incubated for 90 min at 55 ° C. Following hybridization, the coverslips were removed by submerging the slides into approximately 20 ml/slide pre-warmed 25 mM Tris, pH 10, 137 mM NaCl , 3 mM KCl in a water bath at 55° C. and washed for 30 min. Each smear was finally mounted using one drop of Mounting medium (AdvanDx, Woburn, Mass.) and covered with a coverslip.
• Mounting medium AdvancedDx, Woburn, Mass.
• the table displays species identification in the left column, PNA FISH results in the center column, and clinical incidence of the species in the right column.
• three of the species tested gave a positive result, the C. albicans sample contained green fluorescent buds, and the C. glabrata and C. krusei samples contained red fluorescent buds.
• the C. parapsilosis slide had cells which displayed a weak yellow fluorescence which was scored negative.
• the positive results demonstrate that the probe mixture tested produces green signals, indicating fluconazole sensitivity, with C. albicans and red signals, indicating fluconazole resistance, with either C. glabrata or C. krusei . Though only three species are detected with this assay, the species detected represent nearly 70% of the Candida seen clinically in the United States, Europe, Canada, and Latin America.
• This probe mixture and technique would be useful for selection of therapy since a green signal indicates a species ( C. albicans ) which is generally regarded as fluconazole sensitive, whereas a red signal indicates species ( C. glabrata or C. krusei ) which are often or likely fluconazole resistant.
• Example 2 was performed exactly as Example 1, except that a second probe was added to the Fluconazole Sensitive probe set.
• Probe B-Tam Tam-OO-GCAAGGGGCGCAAA-NH 2 (Seq. Id. No.2)
• 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 fluorescent buds. Results are recorded in Table 3. TABLE 3 Species Id. Result Incidence 1 C. albicans Positive/Green (Fluconazole Sensitive) 54.5% C. glabrata Positive/Red (/Fluconazole Resistant) 12.3% C. krusei Positive/Red (/Fluconazole Resistant) 1.5% C.
• Candida spp. was represented by C. guillermondii , C. kefyr , and C. lusitaniae which all have low prevalence in the clinical setting.
• the table displays species identification in the left column, PNA FISH results in the center column, and clinical incidence of the species in the right column.
• the C. albicans and C. parapsilosis samples contained green fluorescent buds, and the C. glabrata and C. krusei samples contained red fluorescent buds.
• the positive results demonstrate that the probe mixture tested produces signals of green, for Fluconazole Sensitive, with C. albicans and C. parapsilosis , and red, for Fluconazole Resistant, with either C. glabrata or C. krusei . Though only four species are detected with this assay, those species represent over 86% of the blood stream infections caused by Candida species in the United States, Europe, Canada, and Latin America.
• This probe mixture and technique would be useful for selection of therapy since a green signal indicates species ( C. albicans and C. parapsilosis ) which are generally regarded as fluconazole sensitive, whereas a red signal indicates species ( C. glabrata or C. krusei ) which are often or likely fluconazole resistant. Neither of the fluorescent signal types (red or green) in this assay positively identifies organisms to a species level; they indicate fluconazole susceptibility-types.
• C. tropicalis is an example of the “susceptible/dose-dependant” susceptibility-type for fluconazole.
• a prospective probe set of a third color specific to the fluconazole susceptible/dose-dependant susceptibility-type can be envisioned, for example, a probe set containing Probe-N. Addition of a prospective third probe set for detection of this susceptibility-type, including C. tropicalis , could account for greater than 96% of the blood stream infections caused by Candida species in the United States, Europe, Canada, and Latin America.
• Example 3 was performed exactly as Example 1, but with a different probe set.
• 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 4 Species Id. Result Incidence 1 C. albicans Negative 54.5% C. glabrata Negative 12.3% C. krusei Negative 1.5% C. parapsilosis Negative 17.8% C. tropicalis Positive/Red (Fluconazole Intermediate) 9.5% Other Candida spp. 2 Negative 4.5% S. epidermidis Negative N/A 1 Average incidence of Candida species for the United States, Europe, Canada, and Latin America. M. A.
• Candida spp. was represented by C. guilliermondii , C. kefyr , and C. lusitaniae which all have low prevalence in the clinical setting.
• N/A Not applicable (Non- Candida species used as controls)
• the table displays species identification in the left column, PNA FISH results in the center column, and clinical incidence of the species in the right column.
• only one of the species tested gave a positive result.
• the C. tropicalis sample contained red fluorescent buds.
• the positive results demonstrate that the probe mixture tested produces red signal, for Fluconazole Intermediate, only with C. tropicalis cells.
• This probe mixture and technique would be useful for selection of therapy since a red signal indicates a species ( C. tropicalis ) which is fluconazole intermediate.
• C. tropicalis is isolated from patients in only ⁇ 10% of BSI caused by fungi, there is clinical value in the ability to rapidly identify the infection as one which is likely sensitive to high doses of fluconazole.
• Example 4 was performed exactly as Example 1, but with a different probe set.
• Probes S-Flu and T-Flu were added in the hybridization solution at 100 nM each. Microscopic examination was conducted using a fluorescence microscope equipped with a FITC/Texas Red dual band filter set. Caspofungin Susceptible/Dose Dependant (S/DD) fungi were identified by green fluorescent buds. Results are recorded in Table 5. TABLE 5 Species Id. Result Incidence 1 C. albicans Negative 54.5% C. glabrata Negative 12.3% C. krusei Positive/Green (Caspofungin S/DD 3 ) 1.5% C. parapsilosis Positive/Green (Caspofungin S/DD 3 ) 17.8% C. tropicalis Negative 9.5% Other Candida spp.
• N/A Not applicable (Non-Candida species used as controls) 3 In Pfaller, et al. (J Clin Microbiol. 2006 Mar; 44(3): 760-3) species were grouped as “most” susceptible to caspofungin that had MIC 90 values between 0.03 and 0.06 ug/ml, and “significantly less” susceptible to caspofungin that had MIC 90 values ⁇ 0.5 ug/ml. Here we interpret “significantly less” susceptibility as susceptible/dose dependant.
• the table displays species identification in the left column, PNA FISH results in the center column, and clinical incidence of the species in the right column.
• Table 5 only two of the species tested gave a positive result.
• the C. parapsilosis and C. krusei samples contained green fluorescent buds.
• the positive results demonstrate that the probe mixture tested produces green signal, for caspofungin Susceptible/Dose Dependant, only with C. parapsilosis and C. krusei cells.
• This probe mixture and technique would be useful for selection of therapy since a green signal indicates two species ( C. parapsilosis and C. krusei ) which require increased dosage of caspofungin.
• C. parapsilosis and C. krusei are isolated from patients in only ⁇ 19.3% (17.8%+1.5%) of BSI caused by fungi, there is clinical value in the ability to rapidly identify the infection as one which is likely to require higher doses of caspofungin.
• Example 5 is performed exactly as Example 4, but with an expanded probe set.
• Probe W is designed to specifically detect Candida guilliermondii (anamorph of Pichia guilliernondii )
• Probe X is designed to specifically detect Candida lusitaniae (also called Clavispora lusitaniae ). Though both of these species are relatively rare in clinical samples, they also require increased dosage of caspofungin (MIC 90 values ⁇ 0.5 ug/ml).
• Use of the Expanded Caspofungin Susceptible/Dose Dependant Probe Set as described here would allow detection of four species which typically require increased caspofungin dosage with a single probe set. This probe mixture and technique would be useful for selection of therapy since a green signal should indicate any of four species ( C. parapsilosis, C. krusei, C. guilliermondii , and C. Iusitaniae ) which require increased dosage of caspofungin.

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