MYCOFAST® EvolutioN 3

Intended use

Mycoplasmas are the smallest and simplest of the procaryotypes capable of self-reproduction (0.15 to 0.25 μm). They differ from other bacteria in their lack of a cell wall and hence a natural resistance to ß-lactams. Since mycoplasmas are relatively fragile, they will only grow in acellular cultures in the presence of various growth factors and at a constant temperature of 35 to 37 °C.

Most human mycoplasmas are commensal. Of the 9 species that have been isolated from the urogenital tract, Ureaplasma urealyticum and Mycoplasma hominis are the most commonly found. U.u. and M.h. are sexually transmitted and can be pathogenic. Respiratory infections or meningitis can occur in the neonate as a result of contamination from the genital tract at birth. In adults, the infections caused by U.u. and M.h. are described in the table below:

Conventional diagnosis is based upon culture on A7 agar plates followed by microscopical identification of U.u. (sea urchin shaped) or M.h. (fried-egg shaped) colonies.
Since both U.u. and M.h. are commensal, infection can only be diagnosed through the determination of the pathological threshold, followed by precise enumeration.

MYCOFAST® EvolutioN 3 gives the solution and makes urogenital diagnosis much easier.

Principle

MYCOFAST® EvolutioN 3 identifies U.u. and M.h. growth after a 24 hour incubation in a liquid medium. During growth, U.u. and M.h. metabolize urea and arginine respectively resulting in a color change of the medium, which contains phenol red indicator, from yellow-orange to red. This color change is due to liberation of ammonia resulting in an alkaline pH of the medium.

Mycoplasma growth thus viewed enables:

• Enumeration of mycoplasma based on the rate of urea or arginine hydrolysis, which is proportional to the number of germs contained in the sample.
  U.u:  > 10³, 10⁴ and 10⁵ UCC/ml
  M.h: > 10⁴ UCC/ml
• Identification based on the sensitivity or otherwise of the germ to three antibiotics (lincomycin, SXT and erythromycin) =  Identibiotic.
• U.u and M.h susceptibility testing to 7 antibiotics:
  Doxycycline (DO) 4-8 μg/mL ,  Pristinamycin (PT) 2 μg/mL , Roxithromycin (ROX) 1-4 μg/mL, Azithromycin (AZM) 0.5-4 μg/mL, Josamycin (JM) 1-4 μg/mL, Ciprofloxacin (CIP) 1-2 μg/mL et Ofloxacin (OFX) 1-4 μg/mL.

Simple protocol

Easy-to-read and easy-to-interpret results

The results are read by the color obtained in the different wells. Urogenital mycoplasma growth is indicated when the medium turns red (alkaline). The medium remains yellow when no growth of urogenital mycoplasma occurs.

Reagents and material

• UMMt:
  • Vial of mycoplasma transport medium (3 mL)
  • Quantity: 25
• UMMlyo:
  • Vial of lyophilized growth medium (+3 mL UMMt)
  • Quantity: 25
• MYCOFAST® EvolutioN 3 trays:
  • Tray of 20-well wrapped individually in an aluminum sachet
  • Quantity: 25
• S.M.h:
  • Mycoplama hominis growth activator (4.5 mL)
  • Quantity: 2

Stability and storage

• All the reagents are ready-to-use. The vials may be stored at 2-8 °C, in their original packaging until the expiry date shown on the kit.
• The UMMt medium may be stored temporarily at room temperature but is more stable at 2-8 °C.

Performance
1 – Detection, identification and enumeration

• • A comparative study with reference strains and clinical strains was carried out between the A7 Agar solid medium method and the MYCOFAST® EvolutioN 3 liquid medium method. The U. urealyticum (n=29) and M. hominis (n=44) strains were tested separately or as a mixture at different dilutions from 103 to >106 CFU/mL on A7 Agar.
  • For enumeration the overall concordance between the two methods is 88.4% (97.8% for a concordance to within one dilution).
  • For identification the overall concordance is 97% for the Identibiotique. The very high concentrations
    (>106 CFU/mL) of U. urealyticum were capable of causing a color change in well 6 (E).

2 – Antimicrobial susceptibility testing

• A comparative study was carried out between the method to determine the minimal inhibitory concentrations (MIC) in a liquid medium and the MYCOFAST® Evolution 3 method. The strains tested were reference strains, clinical wild-type strains, or strains having acquired resistance. Each strain was tested at dilutions of 103, 104 and 105 CCU/mL. The results of the two methods were interpreted as being Sensitive (S), Intermediate (I) or Resistant (R), according to the recommendations of the “Comité de l’Antibiogramme de la Société Française de Microbiologie” (CA-SFM), and are represented in the table below.

Ureaplasma urealyticum (n = 30)
Results of 6 wild-type strains, 3 strains resistant to tetracyclines and 2 strains resistant to fluoroquinolones:

Concordant test

DO: 27
PT: 30
ROX: 24
AZM: 30
JM: 30
CIP: 23
OFX: 7

md

DO: 3
PT: –
ROX: 6
AZM: –
JM: –
CIP: 7
OFX: 22*

MD

DO: –
PT: –
ROX: –
AZM: –
JM: –
CIP: –
OFX: 1

Mycoplasma hominis (n=30)
Results of 6 wild-type strains, 2 strains resistant to tetracyclines, 2 strains resistant to josamycin and 3 strains resistant to fluoroquinolones:

Concordant test

DO: 28
PT: 30
ROX: 30
AZM: 30
JM: 30
CIP: 25
OFX: 25

md

DO: –
PT: –
ROX: –
AZM: –
JM: –
CIP: 2
OFX: 5

MD

DO: 2
PT: –
ROX: –
AZM: –
JM: –
CIP: 3
OFX: –

The overall concordance for U. urealyticum is 81.4% (171/210). There are no very major discordances.
* Note: For ofloxacin, the majority of sensitive strains tested exhibited an MIC of 1 μg/mL; equal to the lower critical concentration.

The overall concordance for M. hominis is 94.3% (198/210). There are no very major discordances.
md: minor discordance, MD: major discordance
Material required but not provided

• Sample collecting material (Swabs, cytobrushes, sterile containers for liquid samples)
• Pipettes and tips
• Waste container for contaminated waste
• Paraffin oil
• Incubator at 37 ± 1 °C