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Histoplasma Capsulatum Culture Identification Test

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Histoplasma Capsulatum Culture Identification Test PRINCIPLES OF THE PROCEDURE Nucleic acid hybridization tests are based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes (3). The ACCUPROBE SYSTEM uses a single-stranded DNA probe with a chemiluminescent label that is complementary to the ribosomal RNA of the target organism. After the ribosomal RNA is released from the organism, the labeled DNA probe combines with the target organism's ribosomal RNA to form a stable DNA:RNA hybrid. The Selection Reagent allows for the differentiation of non-hybridized and hybridized probe. The labeled DNA:RNA hybrids are measured in a Gen-Probe luminometer. A positive result is a luminometer reading equal to or greater than the HISTOPLASMA CAPSULATUM cut-off. A value less than this cut-off is a negative result. CULTURE IDENTIFICATION TEST REAGENTS Reagents for the ACCUPROBE HISTOPLASMA CAPSULATUM INTENDED USE CULTURE IDENTIFICATION TEST are provided in three separate reagent kits: The ACCUPROBE HISTOPLASMA CAPSULATUM CULTURE IDENTIFICATION TEST is a rapid DNA probe test which utilizes the ACCUPROBE HISTOPLASMA CAPSULATUM PROBE KIT technique of nucleic acid hybridization for the identification of Probe Reagent (10 x 2 tubes). Histoplasma capsulatum isolated from culture. Histoplasma capsulatum. Lysing Reagent (1 x 20 tubes). SUMMARY AND EXPLANATION OF THE TEST Glass beads and buffer. Histoplasma capsulatum (H. capsulatum) is the causative agent of the systemic fungal disease histoplasmosis. The organism occurs ACCUPROBE CULTURE IDENTIFICATION REAGENT KIT throughout the world, but is most commonly found in soil from the Reagent 1 (Lysis Reagent). 1 x 10 mL fertile river valleys of North America. Large numbers of infectious buffered solution containing 0.04% sodium azide. conidia are especially associated with areas containing bird or bat Reagent 2 (Hybridization Buffer). 1 x 10 mL excrement. (6, 10) The organism is also found in two other varieties: buffered solution. H. capsulatum var. duboisii (African histoplasmosis) and H. capsulatum var. farciminosum (epizootic lymphaginitis of horses and Reagent 3 (Selection Reagent). 1 x 60 mL mules). The teleomorph (mating or sexual) state of H. capsulatum is buffered solution. Ajellomyces capsulatus. (8) H. capsulatum is a dimorphic fungus. The organism grows as a GEN-PROBE DETECTION REAGENT KIT filamentous fungus under appropriate soil conditions or on room Detection Reagent I. 1 x 240 mL temperature fungal media. In mammalian cells or on specialized 0.1% hydrogen peroxide in 0.001 N nitric acid. fungal media at 37°C, the organism grows as a small budding yeast. Detection Reagent II. 1 x 240 mL Conventional laboratory identification methods begin with culture of the 1 N sodium hydroxide. clinical specimen at room temperature on specialized fungal media, which isolates the slower growing H. capsulatum colonies from possible contaminants such as bacteria and faster growing saprobic WARNINGS AND PRECAUTIONS fungi. Growth of H. capsulatum to a visible colony may take from two A. For in vitro diagnostic use. to four weeks or longer. (4, 8) Some clinical laboratories hold their cultures for as long as twelve weeks before reporting them as B. Use universal precautions when performing this assay (1). negative. (7) C. Use only for the determination of H. capsulatum isolated from Additional growth is needed before the characteristic colony culture. morphology and microscopic sporulation pattern with tuberculate macroconidia may be demonstrated. About 10% of cultures produce D. Use only supplied or specified disposable laboratory ware. only smooth walled macroconidia. (8) Some cultures fail to sporulate. E. Culture handling and all procedural steps through the heat (4, 5) Unusual glabrous, brown or gray colonies are sometimes inactivation step should be performed in a Class II Biological encountered. Many species of fungi other than H. capsulatum Safety Cabinet. produce similar colony and sporulation characteristics. Some examples are Blastomyces dermatitidis, Chrysosporium sp., and F. Reagents in this kit contain sodium azide which may react with Sepedonium sp. (4, 8) Therefore, additional testing is needed to lead or copper plumbing to form potentially explosive metal definitively identify the organism. One method is to convert the fungus azides. Upon disposal of these reagents, always dilute the colony from the filamentous, mycelial phase to the yeast phase by material with a large volume of water to prevent azide buildup in subculture to highly enriched cysteine-containing media and incubation the plumbing. at 35° to 37°C. Conversion to the yeast form is often difficult and may require several transfers at three-day intervals.(4) Other confirmatory G. Avoid contact of Detection Reagents I and II with skin and identification methods based on exoantigen extraction have been mucous membranes. Wash with water if these reagents come described, which take from three to five days or longer to perform. (2, into contact with skin. If spills of these reagents occur, dilute with 4, 9). water before wiping dry. The ACCUPROBE HISTOPLASMA CAPSULATUM CULTURE IDENTIFICATION TEST identifies both phases regardless of STORAGE AND HANDLING REQUIREMENTS sporulation in less than an hour based on identification of specific Probe Reagent Tubes must be stored in the foil pouches at 2° to 8°C. ribosomal RNA sequences that are unique to H. capsulatum. The Probe Reagent Tubes are stable in the unopened pouches until the expiration date indicated. Once opened, the pouch should be resealed and the tubes should be used within two months and prior to the expiration date. 1 102962 Rev. K Other reagents used in the ACCUPROBE HISTOPLASMA b. Run the sonicator for 15 minutes to thoroughly degas CAPSULATUM CULTURE IDENTIFICATION TEST may be stored the water. between 2° to 25°C and are stable until the expiration date indicated. 2. Adjust one heating block or water bath to 60° ± 1°C and DO NOT FREEZE THE REAGENTS . another heating block or water bath to 95° ± 5°C. 3. Prepare the GEN-PROBE luminometer for operation. Make sure there is sufficient volume of Detection Reagents I SAMPLE COLLECTION AND PREPARATION and II to complete the tests. The ACCUPROBE HISTOPLASMA CAPSULATUM CULTURE B. CONTROLS IDENTIFICATION TEST is designed to determine the identity of H. capsulatum isolated from culture. Colonies may be tested as soon as Positive and negative control strains should be tested routinely in growth is visible. Yeast colonies should be no more than one week each laboratory according to local regulations. A culture of H. old from the time growth is first observed. Filamentous colonies capsulatum (e.g., American Type Culture Collection, ATCC should be no more than one month old from the time growth is first #38904) may be used as the positive control, while a culture of observed. Sporulation is not necessary. Blastomyces dermatitidis (e.g., ATCC #60916) may be used as the negative control. A. Solid Media Method. Growth from appropriate solid media such as Sabouraud Dextrose, Brain Heart Infusion, Mycobiotic C. SAMPLE PREPARATION (Mycosel), Inhibitory Mold Agar, Cottonseed Agar, Corn Meal 1. Label a sufficient number of Lysing Reagent Tubes to test Agar, Yeast Nitrogen Base Agar or 5% Sheep Blood Agar the culture isolates and/or controls. Remove and retain the suggestive of H. capsulatum may be tested. caps. 1. Growth can be removed with a 1 µL disposable plastic loop, 2. Pipette 100 µL of Reagent 1 (Lysis Reagent) and 100 µL of a wire loop, a disposable plastic needle, or an applicator Reagent 2 (Hybridization Buffer) into all Lysing Reagent stick. Swabs should not be used due to the small volume of Tubes. If broth cultures are to be tested, do not add liquid in which the cells are subsequently resuspended. Reagent 1 to the Lysing Reagent Tubes. 2. Avoid taking large amounts of the solid media with the cells. 3. Transfer the sample from the solid media or 100 µL of a 2 well mixed broth culture into the labeled Lysing Reagent 3. A 1-2 mm size sample of yeast or filamentous growth is Tubes as described in the SAMPLE COLLECTION AND recommended for the test. PREPARATION Section. Twirl the loop, needle or stick in 4. The operator may elect to inoculate another culture plate at the Reagent 1 and Reagent 2 diluent mixture to remove the this time to confirm the purity of the isolate. cells if testing growth from solid media. B. Broth Culture Method. Growth in Brain Heart Infusion broth with 4. Recap the Lysing Reagent Tubes and briefly VORTEX. turbidity equivalent to or greater than a McFarland 1 D. SAMPLE LYSIS Nephelometer Standard may be tested with the ACCUPROBE 1. Push the Lysing Reagent Tubes through the Sonicator Rack HISTOPLASMA CAPSULATUM CULTURE IDENTIFICATION TEST. Pipette a 100 µL sample from the well-mixed broth so that the reaction mixture in the bottom of the tube is suspension into the Lysing Reagent Tube as described below. submerged but the caps are above the water. Place Sonicator Rack on water bath sonicator. DO NOT ALLOW THE TUBES TO TOUCH THE BOTTOM OR SIDES OF THE MATERIALS PROVIDED SONICATOR. The ACCUPROBE® HISTOPLASMA CAPSULATUM CULTURE 2. Sonicate for 15 minutes. IDENTIFICATION TEST 3. Place the Lysing Reagent Tubes, containing the sonicated organisms in a heating block or water bath for 10 minutes at Cat. No. 2910 20 Tests 95° ± 5°C. Probe Reagent 10 x 2 tubes Lysing Reagent 1 x 20 tubes 4. Carefully remove the Lysing Reagent Tubes from the heating block or water bath. MATERIALS REQUIRED BUT NOT PROVIDED E. HYBRIDIZATION 1 µL plastic sterile inoculating loops, wire loops, plastic needles, 1. Open the foil pouch by cutting evenly across the top of the or applicator sticks for selecting colonies. pouch. Remove enough Probe Reagent Tubes to test the Control culture strains culture isolates and/or controls. Reseal the pouch by folding Water bath or heating block (60° ± 1° C) the opened edge over several times and securing with Water bath or heating block (95° ± 5° C) adhesive tape or a clip.
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