Serology MUG

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.

Given their lack of immunogenicity, serological testing of mycoplasmas does not replace direct diagnosis from patient specimens. The serologic test for urogenital mycoplasmas is especially used to diagnose a deep genital infection, like salpingitis or prostatitis.
The technique of metabolic inhibition using lyophilized reagents and reference strains is the most appropriate technique for use by non-specialized laboratories.

Simple protocol

The quantitative detection of anti-U.u. and/or anti-M.h. antibodies is achieved by a metabolic inhibition technique. The reaction is carried out in the wells of a tray containing in the U. urealyticum rows, lincomycin and in the M. hominis rows, erythromycin. The first 8 wells, of each side of the tray, are inoculated with UMM culture medium containing a pH indicator and seeded beforehand with a known quantity of U.u. and M.h. strains.

The serum to be tested is serially diluted in the tray. The presence of antibodies in the serum causes an inhibition of mycoplasma multiplication and is visualized by a yellow coloration of the medium. However, in the absence of antibodies the mycoplasmas multiply, degrading urea and/or arginine present in the medium, which results in an orangey, red or fuschia coloration of the medium.

For each row of U.u. and M.h. tests there is a growth control. These are carried out in order to verify the viability of the U.u. and M.h. strains, as well as the correct operation of the test.

Easy-to-read and eay-to-interpret results

The presence of antibodies is determined by the absence of a color change of the medium. In the absence of antibodies the medium becomes orangey-red or fuschia colored. The strongest inhibited dilution (the last yellow well) represents the titre of antibodies (see previous scheme).

Reagents and Material

• UMMt:
  • Vial of 2 mL of liquid mycoplasma base medium
  • Quantty: 14
• UMMlyo:
  • Vial of lyophilized mycoplasma growth medium (+ 2 mL UMMt)
  • Quantity: 14
• U.u/M.h:
  • Vial containing lyophilized U. urealyticum and M. hominis strains
  • Quantity: 14
• SEROLOGY tray:
  • 20-well tray wrapped individually in an aluminum sachet
  • Quantity: 14
• Positive Control:
  • Vial containing a lyophilized solution of known titre for verification of the reagents
  • Quantity: 1

Stability and Storage
The reagents, stored at 2 to 8 °C in their original packaging, are stable until the expiry date shown on the kit.
Performance
A comparative study was carried out with the SEROLOGY reagent and the MYCOKIT SERO reagent of PBS ORGENICS on 100 routinely tested human serum samples (64 serum samples negative for anti U.u. and anti-M.h. antibodies, 28 serum samples positive for anti- U.u. and anti-M.h. antibodies, 6 serum samples positive for anti-U.u. antibodies and negative for anti-M.h. antibodies, and 2 serum samples negative for anti-U.u. antibodies and positive for anti-M.h. antibodies). Both methods are based upon the same principle of metabolic inhibition. Reading of the SEROLOGY tray was carried out after incubation for 48 hours:

• For the detection of anti-U.u. antibodies, the sensitivity is 88.2% and the specificity is 100%. The 4 false negatives corresponded to a serum sample positive at dilutions of 1/2 and 1/4.
• For the detection of anti-M.h. antibodies, the sensitivity is 96.6% and the specificity is 98.5%.
• For the determination of the antibody titre, the results of the study indicated a concordance of 92.5%, irrespective of the magnitude of the difference in the titre, and a concordance of 97.5% for titres to within one dilution.

Material required but not provided

• Sterile pipettes
• Sterile distilled water
• Paraffin oil
• Incubator calibrated to 37 °C
• Waste container for contaminated waste