Mycoplasma System Plus : Principle , Test procedure and Interpretation of the results

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Mycoplasma System Plus is a 24-well system that contains desiccated biochemical and antibiotic substrates for the detection, semiquantitative determination, and susceptibility testing of urogenital mycoplasmas such as Mycoplasma hominis and Ureaplasma urealyticum. The system is inoculated with the clinical sample suspension and incubated at 36 1°C. After 24 hours of incubation, the test results are read and interpreted by assessing the color change of the various wells and performing a microscopic examination.

Note that, Ureaplasma urealyticum has been split into two distinct species: Ureaplasma parvum and Ureaplasma urealyticum. When using this device, they are grouped together as Ureaplasma spp. and denoted as Uu.

Principle of Mycoplasma System Plus

MycoplasmaSystem Plus detects, semi-quantifies, and presumptively identifies Mycoplasma hominis and Ureaplasma spp., as well as detects and presumptively identifies the microorganisms most commonly isolated from vaginal and urethral swabs and seminal fluid, Trichomonas vaginalis and Candida spp.

  • The semi-quantitative count of Ureaplasma spp. is indicated by a color change from yellow to red in wells 1-Uu 103, 2-Uu 104, and 3-Uu 105.
  • The semi-quantitative count of Mycoplasma hominis is indicated by a color change from yellow to red in wells 4-Mh 104 and 5-Mh 105.
  • The presence of Trichomonas vaginalis and Candida spp. is determined by examining a drop of culture liquid taken from well 6-TR/YE under a microscope (40x) for the presence of mobile ciliated trophozoites for Trichomonas vaginalis and/or the presence of chlamydospores and hyphae for Candida spp.
  • The susceptibility of urogenital mycoplasmas is tested by observing a color change from yellow to red in wells 7-TE to 24-AZM and then following the instructions under INTERPRETATION OF THE RESULTS.

Configuration of Mycoplasma System plus Wells

WellCount and identification of mycoplasmas / ureaplasmas
1-Uu 103Ureaplasma spp. (titre = 103 CFU/mL)
2-Uu 104Ureaplasma spp. (titre = 104 CFU/mL)
3-Uu ≥ 105Ureaplasma spp. (titre ≥ 105  CFU/mL)
4-Mh 104Mycoplasma hominis (titre = 104 CFU/mL)
5-Mh ≥ 105Mycoplasma hominis (titre ≥ 105 CFU/mL)
WellDetection of T. vaginalis and Candida spp.
6-TR/YETrichomonas vaginalis / Candida spp.
WellSusceptibility test of the mycoplasmas / ureaplasmas
7-TETetracycline – 4 µg/mL
8-TETetracycline – 8 µg/mL
9-PEFPefloxacin – 8 µg/mL
10-PEFPefloxacin – 16 µg/mL
11-OFXOfloxacin – 1 µg/mL
12-OFXOfloxacin – 4 µg/mL
13-DODoxycycline – 4 µg/mL
14-DODoxycycline – 8 µg/mL
15-EErythromycin – 8 µg/mL
16-EErythromycin – 16 µg/mL
17-CLAClarithromycin – 8 µg/mL
18-CLAClarithromycin – 16 µg/mL
19-MNMinocycline – 4 µg/mL
20-MNMinocycline – 8 µg/mL
21-CDClindamycin – 4 µg/mL
22-CDClindamycin – 8 µg/mL
23-AZMAzithromycin – 4 µg/mL
24-AZMAzithromycin – 8 µg/mL

Collection and storage of the samples

  • Using synthetic fiber swabs, collect the vaginal or urethral secretion.
  • Obtain the seminal fluid using the method intended for the microbiological culture examination.
  • The samples must be sent for inoculation in the Mycoplasma System Plus as soon as possible after being collected.
  • Even for short periods of time, do not store in the refrigerator because low temperatures can harm the vitality of particularly sensitive microorganisms like Trichomonas vaginalis and alter the final result.

Test procedure

Preparation of the clinical sample

Using the ampoule cracker included with the kit, carefully open a vial of Physiological Solution.

Vaginal swab – urethral swab

After obtaining the clinical material, immerse the swab in the vial of physiological solution for 5 minutes. Squeeze the swab gently against the vial wall to disperse the clinical material evenly in the Physiological Solution.

Take note to keep the swab dipped in nutritive broth until the test is finished.

Seminal fluid

Shake the vial of Physiological Solution* and wait 5 minutes before inoculating with 0.2 mL of sample.

Urine

10 mL of urinary sample should be centrifuged. Collect 0.1 mL of urinary sediment and place it in the kit’s vial of Physiological Solution; shake and wait 5 minutes before inoculating the system.

Clinical material from mycoplasma transport broth

In the vial of Physiological Solution, add 1 mL Mycoplasma Transport Broth containing the sample under examination, shake gently, and wait 5 minutes before inoculation.

Note: Keep the specimen in the vial of Mycoplasma Transport Broth until the test is finished.

Inoculation of the system

  1. Take a system from its wrapper and bring it to room temperature.
  2. Write down the name of the patient, the date of the start of the examination, and the type of clinical material.
  3. Transfer 0.2 mL of suspension of the clinical sample into each well of the system.
  4. Overlay with 1 drop Vaseline oil for microbiological use all the wells except for well 6-TR/YE.
  5. Cover the system with the lid provided and incubate at 36 ± 1°C for 24 hours, and up to 48 hours.
  6. Observe and confirm the presence of mycoplasmas within 48 hours incubation.

Interpretation of the results

Count and Identification of Mycoplasmas / Ureaplasmas

Watch for the color change in the wells 1-Uu 103 to 5-Mh ≥ 105 and interpret the results as shown below.

Detection of Trichomonas vaginalis and Candida spp.

Take a drop of liquid from the well, place it on a glass slide, cover it with a cover slip, and examine it under a 40x microscope for the presence of T. vaginalis and Candida spp. As shown below, interpret as follows.

Antimicrobial Susceptibility Testing of Mycoplasmas / Ureaplasmas

Keep an eye out for a color change in wells 7-TE to 24-AZM and interpret the results as shown below. For each antibiotic, the results of two wells are compared. Wells 1-Uu 103 and 4-Mh 104 are used as growth controls for the antibiograms of Ureaplasma and M. hominis, respectively.

Note the results on the TEST RESULTS FORM (copy as many forms as necessary).

WellCount and identification of Mycoplasmas / ureaplasmasWell colorWell color
Positive reaction*Negative reaction
1-Uu 103Ureaplasma spp. (titre = 103 CFU/mL) (1)Redyellow
2-Uu 104Ureaplasma spp. (titre = 104 CFU/mL) (2)RedYellow
3-Uu ≥ 105Ureaplasma spp. (titre ≥ 105  CFU/mL) (3)RedYellow
4-Mh 104Mycoplasma hominis (titre = 104 CFU/mL)RedYellow
5-Mh ≥ 105Mycoplasma hominis (titre ≥ 105 CFU/mL)RedYellow

CFU: Colony Forming Units

(1) = Equivalent to 5-20 colonies grown on Mycoplasma Agar A7

(2) = Equivalent to 20-50 colonies grown on Mycoplasma Agar A7

(3) = Over 50 colonies grown on Mycoplasma Agar A7

WellT. Vaginalis and candida spp.Microscope examination (40x)
6-TR/YETrichomonas vaginalis / Candida spp.T. vaginalis: mobile ciliated protozoites Candida spp.:chlamydospores and hyphae

Susceptibility testing of mycoplasmas / ureaplasmas

WellsAntibioticConcentrations (µg/Ml)
7-TE and 8-TETetracycline4 and 8
9-PEF and 10-PEFPefloxacin8 and 16
11-OFX and 12-OFXOfloxacin1 and 4
13-DO and 14-DODoxycycline4 and 8
15-E and 16-EErythromycin8 and 16
17-CLA and 18-CLAClarithromycin8 and 16
19-MN and 20-MNMinocycline4 and 8
21-CD and 22-CDClindamycin4 and 8
23-AZM and 24-AZMAzithromycin4 and 8

Well Color

  • A red* coloring indicates growth (positive) at the tested antibiotic concentration.
  • A yellow coloring indicates no growth (negative) at the tested antibiotic concentration.

Color changes in two successive wells containing the same antibiotic are used to categories results into three susceptibility categories:

SensitiveGrowth is inhibited (YELLOW) by the higher and lower critical concentration of the antibiotic.
IntermediateGrowth is inhibited (YELLOW) by the higher critical concentration of the antibiotic,  But not (RED) by the lower critical concentration.
ResistantGrowth is not inhibited (RED) by the higher and lower critical concentration of the antibiotic.

*An orangey coloring should be considered as test positive/growth.

Quality control

Every batch of Mycoplasma System Plus is subjected to quality control using the following reference microorganisms:

Mycoplasma hominisATCC 23114
Ureaplasma urealiticumATCC 27618
Candida albicansATCC 10231

Factors that may invalidate the results

Poor inoculum standardization; inappropriate clinical material; use of expired systems and/or reagents; non-compliance with incubation temperatures and times

Limits and warnings

  • For accurate test results, proper specimen collection and handling techniques are essential. A negative test result does not always imply that there is no infection. A positive Mycoplasma System Plus test result indicates urogenital mycoplasmas infection, but it cannot be used to diagnose the disease.
  • Some bacteria that produce urease and are present in concentrations of 1067 CFU/mL may cause all of the wells in the panel to change color. The presence of these can be confirmed by reisolating from the original broth on agar medium (e.g., chocolate agar) (see TEST PROCEDURE)
  • A negative result for the lower antibiotic concentration and a positive result for the higher antibiotic concentration is insignificant. The test must then be repeated in that case.

Precautions

The product Mycoplasma System Plus is not classified as hazardous under current legislation; however, for proper use, consult the safety data sheet. Mycoplasma System Plus is a disposable device intended solely for in vitro diagnostic use. It must be used in the laboratory by properly trained personnel who follow approved aseptic and safety procedures when handling pathogenic agents.

Storage

Store in the original packaging at 2-8°C. Keep away from heat sources and avoid drastic temperature changes. In such cases, the product will be valid until the expiry date specified on the label. Do not use after that date. If there are signs of deterioration, discard without using.

Disposal of used material

After use, Mycoplasma System Plus and material that has come into contact with the sample must be decontaminated and disposed of in accordance with the techniques used in the laboratory for decontamination and disposal of potentially infected material.

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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