Keul-o-test

Mycoplama UU-MH

(pipette not supplied)

Dehydrated culture medium based assay for the qualitative detection, indicative enumeration and antibiotic susceptibility testing of UU (Ureaplasma urealyticum) and MH (Mycoplasma hominis) in human genitourinary tract.

Introduction
Mycoplasma is one of the main pathogens which lead to NGU (nongonoccal urethritis), cervical, pelvic inflammatory disease, orchitis, epididymitis etc., and can cause infertility to men and women1. These pathogens can attack and destroy genitourinary epithelial cells, cause infection of the AIDS and other sexually transmitted diseases. Clinical sexually transmitted diseases can be caused by Mycoplasma (mainly by UU and MH). Its occurrence has been presenting an up-trend. Antibiotic resistance is becoming more and more severe due to the misuse of antibiotics, which seriously endanger mankind's health2. The key to the treatment and prevention of the spread of Mycoplasma is timely and accurate diagnosis. Mycoplasma cultivation is currently still being recognized as a reliable method to diagnose the Mycoplasma infection.

Measurement Principle
Mycoplasma kit is based on cultivation and biochemical reactions. The mixed medium is prepared by mixing the freeze-dried powder and the diluent. After Mycoplasma has been cultivated, urea can be decomposed by urease in UU and release NH₃ ³; and arginine can be decomposed by arginase in MH and release NH₃. NH₃ increases the pH of the liquid medium, the result is judged according to the color change of the indicator. Strip contains 12 antibiotics and each one has two concentrations, If Mycoplasma is sensitive to antibiotic, the activity of enzyme is inhibited, so there is no change in color.

Components

1. Strip
   strips each containing 30 wells, among which the UU well is coated with lincomycin, MH well is coated with erythrocin,UU≥104 well is coated with lincomycin and inhibition agent, MH≥104 well is coated with erythrocin and inhibition agent and MIN wells are coated with mincycline at the concentration of 2/8 mg/l, DOX wells are coated with doxycycline at the concentration of 4/8 mg/l, ERY wells are coated with erythromycinat the concentration of 2/8 mg/l, AZI wells are coated with azithromycin at the concentration of 1/4 mg/l, JOS wells are coated with josamycin at the concentration of 2/8 mg/l, THI wells are coated with thiamphenicol at the concentration of 2/8 mg/l, CLI wells are coated with clindamycin at the concentration of 1/4 mg/l, CLA wells are coated with clarithromycin at the concentration of 1/4 mg/l, ROX wells are coated with roxithromycinat the concentration of 1/4 mg/l, SPA wells are coated with sparfloxacin at the concentration of 1/4 mg/l, LEV wells are coated with levofloxacin at the concentration of 1/4 mg/l, GAT wells are coated with gatifloxacin at the concentration of 1/4 mg/l.
2. Freeze-dried Powder
   vials each containing 1.2 ml of peptone of bovine origin and beef heart infusion.
3. Diluent
   vials each containing 4 ml of solution which is used to dissolve the freeze-dried powder. The culture medium, after the diluent and freeze-dried powder are mixed together, conforms to the formula which is, of each 1000 ml of purified water, there are 7.27 g of peptone of bovine origin, 2.51 g of yeast extract, 6.55 g of beef heart infusion, 1.45 g of urea, 1.45 g of arginine hydrochloride, 7.9 g of salt-mixture, 182 ml of horse serum, 0.03 g of phenol red, 9.2 ml of growth factors mixture and 9.2 ml of antibiotic mixture. The pH is 6.3 ±0.3.
4. Mineral Oil
   vial containing 28 ml of liquid paraffin.
5. copy of instruction for use
6. sheets of result paper
7. pipettes tips

1. sample collection swabs
2. bacteriology incubator (36 °C,37 °C,38 °C)

Warnings and Precautions

1. For professional use only.
2. Follow the instruction for use carefully. Reliability of assay results cannot be guaranteed if there are any deviations from the instructions in this package insert.
3. Refer to the material safety data sheet and product labeling for any chemical hazards that may be present in this assay.
4. Wear disposable gloves when dealing with samples and reagents.
   Wash hands after operations.
5. Conduct the assay away from bad ambient conditions. e.g. ambient air containing strong acid, strong alkali or volatile gas and so on.
6. The growth of Mycoplasma in the culture broth would not generate turbidity. This assay has adopted a unique method to effectively inhibit the growth of irrelevant bacteria. If the mixed medium occasionally displays turbidity and turns red, this does not indicate a positive result.
7. After adding the mixed medium with added samples to each susceptibility well, if it is observed that the color of the mixed medium in all the other strip wells except the C- well evidently becomes darker or turns to light red, this may be due to the biased alkalinity of samples from patients under pathological conditions. In this case, it is recommended to retest secretion samples from the patients.
8. When testing the antibiotic susceptibility of positive samples validated by normal growth medium, add 50 μl of the cultured positive sample to the mixed medium from this kit and follow the assay procedures mentioned below except that the Mycoplasma must be reinoculated when the bottom of the vial turns red.
   Otherwise the pH increases, the Mycoplasma die very rapidly and the successful reinoculation rate would be low.
9. Consider the samples, reagent vials and strips for testing as potentially infectious material and deal them in accordance with biosafety laboratory practices.
10. Do not use reagents after expiry date.
11. Do not mix or use components from kits with different batch codes.
12. Do not use vials with turbid appearance.
13. Do not use strips which have been damaged: cupules deformed, desiccant sachet open，and aluminum foiled pouch broken.
14. The performance data presented were obtained using the procedure indicated in this package insert. Any change or modification in the procedure may affect the results.
15. Since Mycoplasma have a high affinity for mucus cell membranes, it is important to thoroughly scrape the mucosa so as to collect as many cells as possible.
16. Collect the sample before administering any antibiotic treatment.
17. A standardized technique must be used to prevent contamination by other microorganisms.
18. A sample cannot be considered as negative before 24 hours of incubation
19. If the sample titer is low, the strip cupules may not change color or the color change may be inconsistent.
20. Enumeration in the tests carried out on the strip can only give an indication of the titer. The exact titer can be determined on agar.
21. The antibiotics are tested on the sample, without taking into account the Mycoplasma titer of the sample. In the case of low titers, the real susceptibility of the strain may be different form the result obtained with the strip.
22. A result which is negative at the lowest concentration of an antibiotic, and positive at the highest concentration is meaningless.
    In this case, perform the test again.

Storage

1. Store all components at 2-8 °C.
2. Use the strip within 8 hours once unwrapped.
3. Use the culture medium, after the diluent and freeze-dried powder are mixed together within 72 hours.
4. Use the inoculated medium within 8 hours at 18-28 °C or within 48 hours at 2-8 °C.
5. The mineral oil may be used until the labeled expiry date once opened.
6. During transportation, store the kit at a low temperature and away from sunlight. Ship the kit with ice cubes from May to October.

Sample

1. For males, collect sample from midstream urine with a disposable collection cup or from urethral secretions (expressed prostatic secretion or semen) with a sterile swab.
2. For females, collect sample from cervical secretions or vaginal secretions with a sterile swab.
3. If the sample is to be inoculated to the mixed medium, inoculate within 4 hours. For a special case, store the sample at 2-8 °C and inoculate within 24 hours.
4. If the sample is to be collected and transported with an UTM swab, store the UTM swab sample at room temperature (18-25 °C) within 24 hours, for longer storage, the UTM sample should be stored at 2-8 °C up to 48 hours.
5. If the sample is to be inoculated to the diluent (the inoculated diluent can be used as transported medium), store the inoculated diluent at room temperature (18-25 °C) within 24 hours, for longer storage, the inoculated diluent should be stored at 2-8 °C up to 48 hours.

Reagent Preparation

1. Bring all reagents to room temperature (18-25 °C) prior to use.
2. Adjust the incubator at 37 °C.

Measurement Procedure

1. If the Sample is Collected and Inoculated in the Diluent
   i. At the place of sample collection, add the swab sample or 500 μl of the midstream urine sample to the diluent. Then send the inoculated diluent to the place where the test is to be conducted.
   ii. Add the inoculated diluent to the freeze-dried powder. Place the lid on the vial, and shake to mix completely.
   iii. Add 100 μl of the inoculated medium to all the wells on the strip except C- well. Shake gently to dissolve the coated materials.
   iv. Add 1 drop of mineral oil to each well except C- well.
   v. Cover the strip. Incubate at 36-38 °C for 24 hours.
2. 2. If the Sample is Collected and Inoculated in the UTM Swab
   i. Add the diluent completely to the freeze-dried powder. Shake on a vortex to ensure the pellet is completely dissolved.
   ii. Add 100 μl of the mixed medium to C- well.
   iii. Inoculate 400 μl of the UTM swab sample to the mixed medium.
   Place the lid on the vial, and shake to mix completely.
   iv. Add 100 μl of the inoculated medium to all the wells on the strip except C- well. Shake gently to dissolve the coated materials.
   v. Add 1 drop of mineral oil to each well except C- well.
   vi. Cover the strip. Incubate at 36-38 °C for 24 hours.

Measurement Results
Read the color change on the strip. If the color turns from orange to red or peachblow, it implicates the growth of Mycoplasma; if the color doesn't change, it could be deemed to be negative or sensitive to antibiotics; seldom, the culturing medium turns light red (i.e. the color does not change evidently) after being cultivated for 24 hours. In this case, it is recommended to extend the culture time by another 12-24 hours.
(Because the patient may be infected by Mycoplasma recently, in the recovery period or under antibiotic treatment such that there is only very little amount of Mycoplasma in the sample or the Mycoplasma is inhibited by antibiotics. Consequently, the color change is not evident.)
Notes: the pathological thresholds usually quoted for U. urealyticum are: ≥10⁴ CCU/ml for an urethral sample, ＜10⁴ CCU/ml in a urine stream or sperm sample. The presence of M. hominis at a threshold ≥10⁴ CCU/ml in an endocervical sample is abnormal.

Control Procedure
The recommended control requirement for this assay is to purchase reference strains (UU (ATCC® 27813) and MH (ATCC^® 15488)) separately.
Culture ATCC^® 27813 in the mixed medium. Incubate until the culture medium turns to light red then perform a subculture to another vial of mixed medium and incubate until culture medium turns to light red. Carry out a 1000 folds dilution of this culture medium with sterile saline solution and add 100 μl to a new vial of mixed medium. Inoculate the strip with this final culture. The result is valid if the color of C+, UU, UU ≥ 104, THI (low concentration) and CLA (low concentration) wells turns from orange to red or peachblow. Test ATCC^® 15488 in the same operation as above. The result is valid if the color of C+, MH, MH≥ 10⁴, ERY (both low and high concentration), AZI (both low and high concentration), THI (low concentration), CLA (both low and high concentration) and ROX (both low and high concentration) wells turns from orange to red or peachblow.

Limitations of the Procedure

1. This assay is intended as an aid for the clinical diagnosis. Conduct this assay in conjunction with clinical examination, patient's medical history and other test results.
2. A very small number of alkaline samples may cause the culture medium turn red directly because this test is based on culture and biochemical reactions and the resulting increase in pH leads to the change in the color of the mixed medium.
3. Since the clinical abuse of antibiotics leads to the emergence of a small number of drug-resistant strains, a very small number of false positive results might be obtained despite the adoption of various antibiotics in the culture broth to inhibit irrelevant bacteria. Hence we recommend confirming the positive samples with a Mycoplasma agar plate whenever practicable.

Performance Characteristics

1. Performance with strains
   For mixed medium, 16 pure Mycoplasma strains inoculated at 3 dilutions as well as 8 mixtures of UU and MH at 2 dilutions were detected positive, whatever the dilution. In addition, 19 interferent strains in urogenital samples at 0.5 McFarland, 100 μl were taken and inoculated. The results are negative.
   For strip, 12 pure Mycoplasma strains inoculated at 3 dilutions as well as 12 mixtures of UU and MH at 2 dilutions were correctly identified by the strip. 18 pure Mycoplasma strains were cultured in the mixed medium until the color turns to light red and then were diluted at the concentrate of 104 CCU/ml and were tested. The corresponding UU≥104 or MH≥104 wells turned to red. 6 pure UU strains at 3 dilutions were tested for 18 times.
   The color of ERY well (low concentration) turned to red for 1 test, the color of THI well (low concentration) turned to red for 3 tests and the color of THI well (both low and high concentration) turned to red for 9 tests, the color of CLI well (low concentration) turned to red for 3 tests and the color of CLI well (both low and high concentration) turned to red for 3 tests, the color of ROX well (low concentration) turned to red for 6 tests and the color of ROX well (both low and high concentration) turned to red for 3 tests, the color of SPA well (low concentration) turned to red for 3 tests, the color of MIN, DOX, AZI, JOS, CLA, LEV, GAT wells (both low and high concentration) remained orange for 18 tests. 3 pure MH strains at 3 dilutions were tested for 9 times. The color of ERY, AZI, CLA and ROX wells (both low and high concentration) turned to red, the color of THI well (low concentration) turned to red for 3 tests and the color of THI well (both low and high concentration) turned to red for 3 tests, the color of SPA well (low concentration) turned to red for 3 tests, the color of LEV well turned to red for 1 test, the color of MIN, DOX, JOS, CLI and GAT wells remained orange for 9 tests.
   
2. Measurement Accuracy by Correlation
   A study was performed where samples were tested using this assay and the bioMerieux^® assay. Data were analyzed and are summarized in the following table.
   

Products	UU	MH	UU-MH	Total
This assay	58	11	17	86
bioMerieux	59	11	17	87

In x2 method, P >0.05, there is no obvious difference between the 2 methods.

Literature References

1. Nunez-Calonge R, Caballero P, Redondo C, et al. Ureaplasma urealyticum reduces motility and induces membrane alterations in human spermatozoa. Hum. Reprod. 1998;13(1O):2756-2761.
2. Rylander M, Hallander HO. In vitro comparison of the activity of doxycycline, tetracycline, erythromycin and a new macrolide, CP 62993, against Mycoplasma pneumoniae, Mycoplasma hominis and Ureaplasma urealyticum. Scand J Infect Dis Suppl. 1988;53:12-17.
3. Murray P. Manual of clinical microbiology. 9th ed. Washington D.C.: ASM Press; 2007.

Manufacturer:

Von-Langen-Weg 10 D-48565 Steinfurt Tel.: 02551/4090  Fax.: 02551/1298 Web: www.biogentechnologies.com

akt. 09.11.2011