ENG MYCO WELL D- ONE MYCO WELL D-ONE System for the presumptive identification and antimicrobial susceptibility test of urogenital mycoplasmas, Gardnerella vaginalis, Trichomonas vaginalis, Candida albicans and Candida spp. 1.INTRODUCTION Mycoplasma hominis and Ureaplasma urealyticum / parvum are the species of the class Mollicutes isolated more frequently from the urogenital tract and considered opportunistic pathogens. It has been demonstrated a direct relationship between the isolation of these microorganisms and certain diseases such as bacterial vaginosis (BV), pelvic inflammatory disease, infections during pregnancy, preterm labor and neonatal infections (1-2) Rapid diagnosis available today may involve the use of expensive tests not available in all hospitals and not useful for determining antimicrobial susceptibility, essential for treatments implementations, epidemiological studies, etc. In the light of this, a system that allows rapidly growing, in addition to antimicrobial susceptibility test in just 18-24 hours, without additional equipment, can be an useful tool in the hands of the microbiologist and clinician. 2. PRINCIPLE System composed of a polypropylene plate containing 32 conical wells for better viewing of the colorimetric reactions that occur as result of the growth of microorganisms in specially formulated media for selective culture of: Mycoplasma hominis, Mycoplasma spp., Ureaplasma urealyticum/parvum. The system allows the presumptive identification of Trichomonas vaginalis, Gardnerella vaginalis, Candida spp. and Candida albicans. The plates contains growth control and indicative wells that permit accelerated growth of other agents, not described in this leaflet, for which diagnosis can be used serological tests, microscopic examination or other conventional methods available in the laboratory, directly from the positive wells of the system. 3. MYCO WELL D-ONE KIT CONTENT 3.1 MYCO WELL D-ONE 10 Identification Panels MYCO WELL D-ONE (REF.MS01283) 10 x 10 ml Sterile physiological Saline solution (REF. MS01304) 10 sterile swabs 3.2 MYCO WELL D-ONE 10 Identification Panel MYCO WELL D-ONE (REF.MS01321) 10 x 10 ml Sterile physiological Saline solution (REF. MS01304) 10 x 2 ml Myco D-One Transport Medium (REF. MS01314) 1 x 25 ml Liquid Sterile Paraffin + 1 dropper (REF. MS01316) 10 Sterile swabs 4. REAGENTS REQUIRED BUT NOT PROVIDED General Laboratory equipment 5. COMPOSITION OF TEST PLATE Well 1: Selective Medium for the isolation of Mycoplasma hominis Well 2: Selective Medium for the isolation of Mycoplasma hominis Well 3:Selective Medium for presumptive identification of Mycoplasma hominis Well 4:Selective Medium for presumptive identification of Mycoplasma spp. Well 5:Selective Medium for growing of CCU 10 4 Ureaplasma urealyticum/parvum Well 6: Selective Medium for growing of CCU 10 5 Ureaplasma urealyticum/parvum Well 7: Selective Medium for presumptive identification of Ureaplasma urealyticum/parvum Well 8: Selective Medium for presumptive identification of Ureaplasma urealyticum/parvum Well 9: Culture medium containing Levofloxacin 1 µg/ml Well 10: Culture medium containing Moxifloxacin 0,25µg/mL
Well 11: Culture medium containing Tetracycline 4 µg/ml Well 12: Culture medium containing Clindamycin 0,25 µg/ml Well 13: Culture medium containing Levofloxacin 2 µg/ml Well 14: Culture medium containing Moxifloxacin 2 µg/ml Well 15: Culture medium containing Tetracycline 1 µg/ml Well 16: Culture medium containing Erythromycin 8 µg/ml Well 17: Culture medium containing Levofloxacin 2 µg/ml Well 18: Culture medium containing Moxifloxacin 0,5 µg/ml Well 19: Culture medium containing Tetracycline 8 µg/ml Well 20: Culture medium containing Clindamycin 0,5 µg/ml Well 21: Culture medium containing Levofloxacin 4 µg/ml Well 22: Culture medium containing Moxifloxacin 4 µg/ml Well 23: Culture medium containing Tetracycline 2 µg/ml Well 24: Culture medium containing Erythromycin 16 µg/ml Well 25: Enriched Culture Medium for growth control of Mycoplasma hominis Well 26: Selective Medium for presumptive identification of Candida albicans Well 27: Selective Medium for presumptive identification of Candida spp. Well 28: Selective Medium for presumptive identification of Candida spp. Well 29: Selective Medium for presumptive identification of Gardnerella vaginalis Well 30: Selective Medium for presumptive identification of Gardnerella vaginalis Well 31: Selective Medium for presumptive identification of Trichomonas vaginalis Well 32: Enriched Culture Medium for growth control of Ureaplasma urealyticum/parvum 6. TEST PROCEDURE 6.1 SAMPLE COLLECTION AND PREPARATION Samples: endocervical exudate, vaginal exudate, urethral exudate, urine, seminal fluid, endotracheal fluids. For the best performance of this system the sample must be taken aseptically according to the methodology implemented in each hospital center and before starting antimicrobial treatment. The sample should be processed immediately after collection. Do not refrigerate or freeze. Protect from light and sun. Results obtained by the use of MYCO WELL D-ONE has to be confirmed by other complementary methods established for each laboratory. endocervical exudate, vaginal exudate, urethral exudate : - Take the sample according to the procedures established in each laboratory - Resuspend the swab with the sample in sterile saline solution supplied in the kit, leave in saline solution for 3-5 minutes, stirring and pressing the swab against the tube walls until obtain an homogeneous suspension. - Add 150 µl of the obtained suspension in each well of the system panel - Add two drops of sterile paraffin in the wells from 1 to 25 and 32 - Incubate at 36 ± 1 ⁰C for 24-48 hours. - All readings are carried out visually - In the case of wells 28 and 31, it is recommended microscopic observation (40 X) Urine-Seminal Liquid - Take the sample according to the procedures established in each laboratory - Resuspend 200 µl of the obtained sample in sterile saline solution supplied in the kit
- Add 150 µl of final suspension obtained in each well of the system - Add two drops of sterile paraffin in the wells from 1 to 25 and 32 - Incubate at 36 ± 1 ⁰ C for 24-48 hours - All readings are carried out visually - In the case of wells 28 and 31, it is recommended microscopic observation (40 X) Endotracheal aspirate - endotracheal exudate - Take with a swab or a sterile Pasteur pipette a portion of the sample obtained according to the procedure established in each laboratory for sample collection. - Resuspend the swab into the vial of sterile saline solution contained in the kit. - Add 150 μl of final suspension obtained in each well of the panel system. - Add two drops of sterile paraffin in the wells from 1 to 25 and 32 - Incubate at 36 ± 1 ⁰ C for 24-48 hours. - All readings are performed visually. - In the case of wells 28 and 31, it is recommended microscopic observation (40 X). Note: Other respiratory specimens or related with associated process can be applied to this system of identification according to the procedure of sample collection and processing established in each laboratory and international standard (3-4). Incubation longer than 48 hours may be carried out on the consideration of the microbiologist. In the case of incubation longer than 48 hours, may occur a decrease in the volume of the wells 28, 30 and 31, indicated for a possible microscopic observation of microbial structures. In such case the colorimetric reading is not affected, however for microscopic observation, it can be added a drop of physiological saline and resuspend the content before preparing the slide. The presence of colour in the wells not affect observation of microbial structures. Formulations and additional supplements of certain wells allow the growth of other Mycoplasma species not herein mentioned. It is recommended that in case microbiologist suspected these agents are presents and want to confirm their presence, incubations have to be made up to 6 days and observe the positivity of well 4. Whenever possible additional tests should be performed as growing in plate (5-6). Where possible the use of traditional methods established for conventional identification of microorganisms which can be identified in this panel are recommended (5). 6.2 USE OF MYCO D-ONE TRANSPORT MEDIUM (REF. MS01321 MYCO WELL D-ONE) If the sample is not analyzed immediately after collection and is inoculated in Myco D-One Transport Medium (Transport medium) proceed as follows: SAMPLE: Vaginal exudate, endocervical exudate and urethral exudates 1. Take the sample with a sterile swab. 2. Open the Transport medium tube. 3. Insert into Myco D-One Transport Medium the sterile swab and let it submerged. 4. Before inoculating the sample in the identification panel, take the swab and press it against the wall of the saline solution vial supplied in the kit, shake carefully in order to obtain an homogeneous suspension. 5. Add 150 µl (3 drops) of the obtained suspension in each well of the MYCO WELL D-ONE plate. 6. Add two drops of sterile paraffin in the wells from 1 to 25 and 32. 7. Incubate at 36 ± 1 ⁰ C for 24-48 hours. 8. All readings are performed visually. 9. In the case of well 28 and 31, a complementary microscopic observation (40X) is recommended.
IN THE CASE OF OTHER SAMPLES, IT IS RECOMMENDED TO READ CAREFULLY THE FOLLOWING ISTRUCTIONS: 1. Respiratory specimens or related with an associated process: take a portion of sample with a sterile swab and realize the same procedure described previously. 2. Liquid samples (urine, seminal liquid etc..): Insert a swab for 10 seconds. 3. Take the swab and put in the transport medium and let it submerged. 4. Before inoculating the sample in the identification panel, take the swab and press it against the wall of the saline solution vial supplied in the kit, shake carefully in order to obtain an homogeneous suspension. 5. Add 150 µl (3 drops) of the obtained solution in each well of the identification panel. 6. Add two drops of sterile paraffin in the wells from 1 to 25 and 32. 7. Incubate at 36 ± 1 ⁰ C for 24-48 hours. 8. All readings are performed visually. 9. In the case of well 28 and 31, a complementary microscopic observation (40X) is recommended. 6.3 OBSERVATIONS 1. Incubations longer than 24-48 hours may be realized according to the consideration of the microbiologist, for a period of 6 days, but the formulations of this kit allow the growth of the most common urogenital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum / parvum ) in 18-48 hours (5-6). 2. In the case of well 4 (Mycoplasma spp.) an evident color change to indicate fermenting mycoplasmas species cannot be observed until 3-6 days depending on species. In case of this kind of longer incubation only the well 4 need to be observed, the presence of color change in the rest of the plate must not be taken in consideration. 3. The positivity of well 4, is given by a color change from RED to YELLOW-transparent color. Any turbidity or presence of sediment is indicative of a microorganism in the sample that does not belong to the genus of Mycoplasma. 4. The color change in well 4 to turbid yellow, within 48 hours is not indicative of the growth of Mycoplasma species. In this case, it is recommended to perform confirmatory tests as the conventional culture or molecular methods (7-8). 5. The Sector of antimicrobial susceptibility test should NOT be considered for Mycoplasma spp. In the section 3 of the identification panel, it is necessary consider that some microorganism as Candida spp. may require 48 hours to their growth therefore may occur a decrease in the volume of the wells. In this case the colorimetric reading is not affected, however for microscopic observation, a drop of physiological saline has to be added, and it is necessary resuspend the contents before preparing the slide sheet. The presence of the colorimetric reaction in the wells does not affect the observation of microbial structures. Wherever possible, it is recommended to use traditional methods established for conventional identification of microorganisms which can be identified by this panel (5). 7. RESULTS INTERPRETATION The results are evidenced by color changes that occur in wells according to reactions due to chemical or chromogenic components contained in specific formulations realized for each microorganism (See The Reaction Table). Note: The microorganisms susceptible to Tetracycline are also to doxycycline and microorganisms susceptible to erythromycin are also to azithromycin (9). Additional microscopic observation is recommended as described in this IFU. In the case of positive samples that have to be confirmed, it is recommended the culture in selective media. To perform this procedure, samples can be stored in Myco D-One Transport Medium or it can be realized a culture from the ID wells of the system.
8. WARNING AND PRECAUTIONS 1. For professional in vitro diagnostic use only. 2. The samples have to be manage as potentially infectious and the test must be performed only by trained personnel for handle clinical and / or microbiological laboratory techniques. 3. Do not open the sealed pouch unless ready to perform the assay. 4. Do not use expired devices. 5. Do not use components from any other type of test kit as a substitute for the components in this kit. 6. Wear protective clothing and disposable gloves while handling the kit reagents and clinical specimens. Wash hands thoroughly after performing the test. 7. Do not smoke, drink or eat in areas where specimens or kit reagents are being handled. 8. Dispose of all specimens and materials used to perform the test as bio-hazardous waste. 9. LIMITATIONS Samples collected after antimicrobial treatment can bring false positive results (4-5). The samples such as vaginal or endocervical exudate, with ph values due to infrequent particular infectious processes can cause nonspecific reactions with positive colorations in a large number of wells of the panel, causing a nonspecific effect with positive staining in the areas dedicated to the identification and antimicrobial susceptibility of urogenital mycoplasmas ( 7-8 ). A sample with a high concentration of color changin units of urogenital mycoplasmas can lead to widespread positivity of identification panel, in this cases it is recommended to dilute the sample (10-12). Myco D-ONE Transport Medium is recommended for the preservation of samples and / or transportation. In this way samples can be stored at 4 ⁰C for 24 hours or frozen at -70 ⁰ C for 8 weeks. However it is recommended to inoculate the sample in the plate as soon as possible because the storage may affect the quality of the sample, the concentration of the organism or the presence of particularly "fastidious" strains. A collection or improper storage may cause sample contamination or microbiological agents are incompatible with the proper functioning of this kit. Read this instructions for use carefully. 10.QUALITY CONTROL The control strains are used for both positive reactions from different wells to check the proper functioning of the media formulations of the different wells before any nonspecific reactions. In order to realize the quality control, it is recommended to use the following reference strains: Non fastidous mycoplasmas Strain ATCC Mycoplasma hominis 23114 Mycoplasma penetrans 55252 Ureaplasma urealyticum 27618 For growth control Ureaplasma urealyticum 33175 For antibiotic susceptibility test determination Ureaplasma parvum 27815 Fastidous or slow-growing mycoplasmas Strain ATCC Mycoplasma genitalium 33530 Mycoplasma fermentans 19989 Mycoplasma hyorhinis * 29052 * The inclusion of strain Mycoplasma hyorhinis (cultivar alpha) was performed to test the ability of the formulation to promote the growth of difficult species (M. genitalium and M. fermentans)
Other bacteria tested: Candida albicans ATCC 10231 Candida tropicalis ATCC 13803 Candida dubliniensis ATCC MYA 646 DSM 13268 Gardnerella vaginalis ATCC 14019 Trichomonas vaginalis ATCC 30001 Escherichia coli ATCC 25922 The strains used for Quality Control have been carefully selected according to the recommendations published for the growth and susceptibility to antimicrobial agents (5-9). Each laboratory should establish its own internal quality controls. 11. STORAGE AND CONSERVATION Store at 2-8 C in its original package. Do not store near heat sources and avoid extreme temperature variations. Under these conditions the product is valid until the expiration date indicated on label. Do not use after this date. Discard if there are signs of deterioration.
REACTION TABLE COLORIMETRIC REACTION POSITIVE NEGATIVE Well 1: Selective Medium for the isolation of Mycoplasma hominis RED YELLOW Well 2: Selective Medium for the isolation of Mycoplasma hominis RED YELLOW Well 3:Selective Medium for presumptive identification of Mycoplasma hominis RED YELLOW Well 4:Selective Medium for presumptive identification of Mycoplasma spp. YELLOW RED Well 5:Selective Medium for growing of CCU 10 4 Ureaplasma urealyticum/parvum RED YELLOW Well 6: Selective Medium for growing of CCU 10 5 Ureaplasma urealyticum/parvum RED YELLOW Well 7: Selective Medium for presumptive identification of Ureaplasma urealyticum/parvum Well 8: Selective Medium for presumptive identification of Ureaplasma urealyticum/parvum RED RED RESISTANT SUSCEPTIBILITY TEST YELLOW YELLOW SUSCEPTIBLE Well 9: Culture medium containing Levofloxacin 1 µg/ml RED YELLOW Well 10: Culture medium containing Moxifloxacin 0,25µg/mL RED YELLOW Well 11: Culture medium containing Tetracycline 4 µg/ml RED YELLOW Well 12: Culture medium containing Clindamycin 0,25 µg/ml RED YELLOW Well 13: Culture medium containing Levofloxacin 2 µg/ml RED YELLOW Well 14: Culture medium containing Moxifloxacin 2 µg/ml RED YELLOW Well 15: Culture medium containing Tetracycline 1 µg/ml RED YELLOW Well 16: Culture medium containing Erythromycin 8 µg/ml RED YELLOW Well 17: Culture medium containing Levofloxacin 2 µg/ml RED YELLOW Well 18: Culture medium containing Moxifloxacin 0,5 µg/ml RED YELLOW Well 19: Culture medium containing Tetracycline 8 µg/ml RED YELLOW Well 20: Culture medium containing Clindamycin 0,5 µg/ml RED YELLOW Well 21: Culture medium containing Levofloxacin 4 µg/ml RED YELLOW Well 22: Culture medium containing Moxifloxacin 4 µg/ml RED YELLOW Well 23: Culture medium containing Tetracycline 2 µg/ml RED YELLOW Well 24: Culture medium containing Erythromycin 16 µg/ml RED YELLOW POSITIVE NEGATIVE Well 25: Enriched Culture Medium for growth control of Mycoplasma hominis RED YELLOW Well 26: Selective Medium for presumptive identification of Candida albicans GREEN NO COLOR Well 27: Selective Medium for presumptive identification of Candida spp. RED NO COLOR Well 28: Selective Medium for presumptive identification of Candida spp. TURBID NO COLOR MICROSCOPIC OBSERVATION (40 X) Chlamydospores and hiphae Well 29: Selective Medium for presumptive identification of Gardnerella vaginalis RED NO COLOR Well 30: Selective Medium for presumptive identification of Gardnerella vaginalis PINK NO COLOR GRAM STAINING gram-variable, pleomorphic bacilli Well 31: Selective Medium for presumptive identification of Trichomonas vaginalis SLIGHT BLUE SLIGHT BLUE Well 32: Enriched Culture Medium for growth control of Ureaplasma urealyticum/parvum MICROSCOPIC OBSERVATION (40 X) mobile ciliated trophozoite POSITIVE RED NEGATIVE YELLOW