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3 - PRINCIPLE
well 10, erythromycin is included to inhibit the growth of U.u. MYCOFAST US identifies U.u. and M.h. growth after a 24 hour incubation in a liquid medium. During growth, U.u. and M.h. metabolize 5 - PRECAUTIONS
urea and arginine respectively resulting in a color change of the medium, • The reagents are intended solely for in vitro use and must be
MYCOFAST® US
which contains phenol red indicator, from yellow-orange to red. This color handled by authorized persons.
Urogenital Mycoplasma Diagnosis
change is due to liberation of ammonia resulting in an alkaline pH of the • The patient samples and inoculated reagents are potentially
infectious; they must be handled with caution, observing universal precautions and the current regulations for this type of product in The enumeration of mycoplasmas based on the rate of urea or arginine the country of use.
hydrolysis, which is proportional to the number of organisms contained in • Handle reagents containing raw materials of animal origin with
30 tests (Cat N° 00050)
the sample. (European patent # 0311541 ; US Patent # 5.091.307). The identification based on the resistance profile to three antibiotics at • Do not use reagents after the expiration date.
• Do not use damaged or poorly stored reagents
- U. urealyticum in liquid medium is resistant to Lincomycin, whereas M. • A positive result with the MYCOFAST method indicates
colonisation by urogenital mycoplasmas, but cannot alone be used 1 - INTENDED USE
- M. hominis in liquid medium is resistant to Erythromycin, whereas U. to make a clinical diagnosis. Clinical diagnosis must be made by a MYCOFAST US has been designed for the detection, enumeration urealyticum is susceptible.
doctor and is a function of the biological results and clinical signs. and identification of Ureaplasma urealyticum (U.u.) and Mycoplasma - U. urealyticum and M. hominis are resistant to antifolates of the hominis (M.h.) in endocervical, urethral, urinary, gastric and sperm Trimethoprim / Sulfamethoxazole.
6 - SAMPLE COLLECTION AND HANDLING
4 - REAGENTS
6.1 Sample collection
Endocervical and vaginal sample collection
2 - INTRODUCTION
Description
Amount Use only a Dacron® or rayon swab or a cytobrush to collect
Mycoplasmas are the smallest and simplest of the procaryotypes UMMt: Vial of mycoplasma transport medium (2 mL)
capable of self-reproducing (0.15 to 0.25 µm) (1). 15 human species The cervix should be carefully cleaned with a swab, to remove have been found to date all belonging to the mollicutae class (derived UMMlyo: Vial of lyophilized growth medium
secretions, before collecting the sample with a new swab. As from: cutis mollis; soft skin). They differ from other bacteria in their lack MYCOFAST US Tray: Tray of 2 x 10 wells
Mycoplasmas adhere strongly to mucous cells, the mucous lining
of a cell wall and hence a natural resistance to ß-lactams, as well as sealed in an aluminium package.
should be well scraped to obtain a rich specimen (2).
by the presence of a membrane rich in sterol obtained through their S. M.h. : Mycoplama hominis growth activator (4.5 mL) 2
Urethral sample collection
adhesion to eukaryotic cells. Since Mycoplasmas are relatively fragile, UMMt medium - Composition in g/L of distilled water:
Clean the meatus and swab or scrape the area to obtain cells.
they will only grow in acellular cultures in the presence of various Mycoplasma broth.20 Sperm, Urine
growth factors and at a constant temperature of 35 to 37 °C. Collect sperm or first micturition (urine) in a sterile tube or bottle.
Most human mycoplasmas are commensal. Of the 9 species that have pH : 6.0 ± 0.2 Gastric secretions
been isolated from the urogenital tract, Ureaplasma urealyticum and Reconstituted medium (UMMt+UMMlyo) -
Collect gastric secretions from neonates by aspirating through a Mycoplasma hominis are the most commonly found. U.u. and M.h. are Composition in g/l of distilled water:
catheter and transferring to a sterile bottle.
sexually transmitted and can be pathogenic. Respiratory infections or Mycoplasma broth.20 6.2 Transport in UMMt medium
meningitis can occur in the neonate as a result of contamination from Foal serum. 200 mL the genital tract at birth (3). In adults, the infections caused by U.u.
Swab samples: Place the swab in a vial of UMMt medium.
and M.h. are described in the table below (6) : Liquid samples: Inoculate a vial of UMMt medium with 200 µL of
U. urealyticum
M. hominis
The inoculated UMMt medium may be kept for 8 hours at room
temperature (18-25 °C) or 16 hours at 2-8 °C.
MYCOFAST US tray
7 - PREPARATION AND STORAGE OF REAGENTS
Each tray consists of 2 tests, each with 10 wells. Each test has 3 parts: • All the reagents are ready to use. Store the vials at 2-8 °C,
in their original packaging until the expiration date shown Enumeration for U.u. between 103 and >105 CCU/mL • The UMMt medium may be stored temporarily at room
U.u. and M.h. identification via resistance temperature but is more stable at 2-8 °C.
profiles to Lincomycin (L), Trimethoprim • Should only half a tray be used, the remaining half may be stored,
Conventional diagnosis is based upon culture on A7 agar plates /Sulfamethoxazole (SXT) and Erythromycin (E) prior to use, for up to 3 weeks at 2-8 °C in its original packaging.
followed by microscopicidentification of U.u. (sea urchin shaped) or 7
Enumeration of M.h. (> 104 CCU/mL • The Mh supplement is stable for 3 months after opening
• Do not freeze the reagents contained in the kit.
Since both U.u. and M.h. are commensal, infection can only be diagnosed through the determinination of the pathological threshold, Specific U.u. enumeration is carried out by including lincomycin in the first 3 wells, which inhibit M.h. growth, (if present). To enumerate M.h. in 8 - MATERIAL REQUIRED BUT NOT PROVIDED
10.2.2 Enumeration (wells 1, 2, 3 and 7)
• As with all organism search methods, the quality of the sample can
• Sample collecting material (swabs, cytobrushes, sterile containers Mark the wells that have turned red and interpret:
influence the test result. A negative test does not necessarily indicate Red color
Interpretation (CCU/mL)
• Pipettes and tips
observed in well #
• A sample with a low mycoplasma load (<103 CCU/mL) may lead to a
• Waste container for contaminated waste
random color change in the different wells in the tray. • Paraffin oil
14 - PERFORMANCE
• Incubator at 37 °C ± 1 °C
Clinical samples were used to test the performance of the MYCOFAST method by comparison with culture on A7 agar plates. The results of 9 - METHOD
Notes
the identification are summarized in the following table: Allow the reagents to reach room temperature (20-30 minutes).
• For males, the interpretation criterion for U. urealyticum is > 104
CCU/mL for an urethral sample; for females the interpretation criterion 9.1 Regeneration of UMMlyo medium
for M.hominis is > 104 CCU/mL for an endocervical sample (5).
Urethral
Transfer each inoculated UMMt medium into a new UMMlyo vial. The • The concentration usually quoted for U. urealyticum in a first urine
UMMlyo medium thus regenerated should display an orange tint. Mix stream, sperm, or an endotracheal specimen is 103 CCU/mL (5).
Prevalence
11 - PARTICULAR CASES
Sensitivity
9.2 Inoculation of the tray
Very high U.u and M.h levels
Specificity
• Identify each series of wells.
The content of all the wells on the tray has turned red. It is recommended • Remove the adhesive film and add the following to the wells of each that the sample be diluted in order to obtain more specific results. In this The MYCOFAST method has also been validated for the detection and
identification of mycoplasmas in samples of neonate gastric secretions • Inoculate a new UMMt vial with 200 µL of the original UMMlyo medium (n=208, prevalence 19.2%) and in parallel on cervico-vaginal samples
stored at 2-8 °C (see § 9.2). Ensure that no color change has taken place from the mother (n=208, prevalence 48.1%). All the strains of on removing the medium from the refrigerator. mycoplasma present on the A7 agar were detected with the • Recover the wells with the adhesive film.
• Regenerate a new UMMlyo vial with the whole inoculated UMMt vial.
Store excess UMMlyo medium in its vial at 2-8 °C for at least 48 • Inoculate a new tray with the medium obtained (§ 9.3)
• Take the dilution (1:10) into account in the interpretation of the 15 - WASTE ELIMINATION
Waste should be disposed of in accordance with the hygiene rules and 9.3 Incubation of the tray
• If necessary, confirm the presence of mycoplasmas on an A7 agar plate current regulations for this kind of product in the country of use.
Incubate tray at 37 °C ± 1 °C for 24 hours. by reisolating from the original UMMlyo medium stored at 2-8 °C (see § For U.u. and M.h enumeration, read the results in 24 hours. Prolong 16 - BIBLIOGRAPHY
the incubation for up to 48 hours to enable strains with a weak 1. BEBEAR C., DE BARBEYRAC B. 1994. Les mycoplasmes, p. 1443-1463.
enzymatic activity to become apparent.
Dans FRENEY J., RENAUD F. , HANSEN W. , BOLLET C. (éd.).“Manuel de Note: Incubation can be continued for up to 72 hours for urine, sperm 12 - QUALITY CONTROL
bactériologie clinique”, 2ème éd., vol.3, Elsevier, Paris .
and gastric secretions if necessary.
Quality control can be carried out from a lyophilized reference strain 2. BOUCAUD-MAITRE Y. et THOINET S. 1993. Analyse des prélèvements
(Ureaplasma urealyticum ATCC 27618).
en bactériologie médicale - 2ème partie : prélèvements génitaux. Feuil. Biol., 34 :
10 - READING AND INTERPRETATION
Prepare a preculture after inoculation of 100µL of the regenerated strain 21-24.
in a vial of reconstituted UMM medium (UMMt+UMMlyo).
3. GENIAUX M. 1994. Microbiologie des MST chez les mineurs de moins de 15
10.1 Validation
Incubate at 37 °C ± 1 °C for 6 hours.
ans. Méd Mal Infect. 24:409-424.
Check that all the wells in the row are clear. A cloudy appearing well Inoculate a new UMM broth reconstituted with 200 µL of the 4. GRATTARD F., SOLEIHAC B., DE BARBEYRAC B., BEBEAR C.,
indicates bacterial contamination. In this case repeat the analysis.
SEFFERT P. and POZZETTO B. 1995. Epidemiologic and molecular
10.2 Reading and interpretation
Inoculate the MYCOFAST US tray and perform the test as indicated in investigations of genital mycoplasmas from women and neonates at delivery.
The results are read by the color obtained in the different wells. these instructions (§9.2, 9.3, and 10).
Pediatr. Infect. Dis. J. , 14 : 853-858.
Urogenital Mycoplasma growth is indicated when the medium turns red Expected result:
5. PEREYRE S., BEBEAR C.M., BEBEAR C. 2001. Les mycoplasmes en
(alkaline). The medium remains yellow when no growth of urogenital pathologie humaine. Revue Française des Laboratoires. Supplément au N°329,
Note: An orange tint should be considered as a positive test (rate 6. TAYLOR-ROBINSON D. 1995. Ureaplasma urealyticum (T-strain
Note: In the event of a lyophilized strain with low ureasic activity, Mycoplasma) and Mycoplasma hominis, p. 1713-1718. Dans MANDELL G. L. , incubation can be continued for up to 18-24 hours. The broth should be BENNET J. E. and DOLIN R. (ed.). Principles and Practices of Infectious 10.2.1 Identification (wells 4, 5 and 6)
inoculated with 20 µL of preculture.
Diseases, 4th ed., vol. 2, Churchill Livingstone, New York.
Identification is made according to the color change of specific wells, as well as the observations in wells 4, 5 and 6 which determine the 13 - LIMITATIONS OF THE PROCEDURE
MYCOFAST® is a trademark of ELITech France SAS • Some bacteria that are present in quantities of >106-7 CFU/mL and
Color in well #
contain urease may cause all the wells in the tray to change color. The Place of manufacture
USA DISTRIBUTOR
4 (L) 5 (SXT) 6 (E)
presence of these can be verified by reisolating on chocolate agar from ELITech France the original UMMlyo medium stored at 2-8 °C (see § 9.2).
459 SOUTH MAIN STREET
Ureaplama urealyticum
• A basic sample pH (pH > 8) may lead the UMM medium to change Allée d’Athènes
LOGAN, UTAH 84321
Mycoplasma hominis
color. Should this occur, dilute the sample (1:10) in fresh UMM medium 83870 SIGNES and interpret the results taking the dilution into account.

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