Method for increasing polymerase chain reaction method sensitivity in diagnosing the cases of urogenital infection in patients with marked exudative inflammation

FIELD: medicine.

SUBSTANCE: method involves administering fluoroquinolone-series antibiotic like Ciproflaxin at a dose of 100-250 mg twice a day during 3-5 days and non-steroid anti-inflammatory preparation of Diclophenac at a dose of 75-150 mg/day during 3-7 days are sequentially introduced in preparing patient to diagnostic examination.

EFFECT: enhanced effectiveness in increasing method sensitivity.

2 tbl

 

Development relates to the field of medicine, in particular to urology, Nephrology, venereology, gynecology, pulmonology, obstetrics, neonatology, ophthalmology, may be used in medical biology that can be applied in veterinary medicine.

Known methods of mechanical removal of inflammatory exudate - cotton swabs, cotton swabs [1], the processing of the urethra sterile saline.

It can be insufficiently sensitive in patients with severe exudative inflammation. Scraping will be complicated and, in addition, this method nepriemlim in the diagnosis of Mycoplasma of the urine samples (with pyelonephritis and prostatitis (urine sample after the massage of the prostate gland).

For the best fence material on the intracellular microorganisms for PCR diagnostics use special cytometry[1, 2, 5].

Clinical methods of reducing the exudative inflammation prior to sampling, suggest mechanical leaching products of inflammation (such as urine), but this usually isn't enough to remove dismissed from the labor army related bacteria [4].

In addition, the known method pharmacologically provocation - prednizolonovuyu test to increase the sensitivity of diagnosis of urogenital infections [4].

There is a method of reducing the negative is osdate concomitant microflora on the sample microbiological samples by adding antibiotics to the environment taken by the material during transportation of samples to the laboratory polymerase chain reaction for reduction bacterial contamination [2]. This method is used in the calculation to mitigate the impact of additional contaminaci microorganisms after specimen collection.

Previously used sample preparation methods did not reduce the effect of mixed infection involved in the inflammation along with Mycoplasma infection, namely contamination of samples extraneous DNA or RNA and the error amplification while [11].

In addition, severe exudative inflammatory reaction, represented by the white blood cells and their lysates, a large amount of mucus greatly complicate the selection of specific DNA of Mycoplasma, hominis and genitalium, therefore the results of the trial in patients with acute bacterial exudative inflammation can give a large number of false-negative results on specific intracellular pathogen[1, 12, 13].

These facts were motivated to use the method of concomitant elimination of the contaminating microflora to take the material for PCR diagnostics through the use of antibacterial agents in combination with anti-inflammatory drugs, eliminating exudative inflammation.

The essence of the invention is to increase the sensitivity of diagnosis of Mycoplasma hominis and Mycoplasma genitalium by polymerase chain reaction in patients with acute exudative (OS the eye) inflammation by eliminating concomitant DNA of bacterial pathogens and elimination of additional pollutant factor - lysates of bacteria in exudative inflammation with concomitant use of antibacterial drugs acting on the accompanying microflora, and anti-inflammatory drugs that reduce exudative inflammation.

The essence of the proposed development lies in the fact that the treatment is carried out until sampling with antibiotics and nonsteroidal anti-inflammatory drugs in the following ratio of ingredients:

the fluoroquinolone antibiotics row at a dose of 100-250 mg 2 times a day, 3-5 days,

- nonsteroidal anti-inflammatory drugs in medium-high therapeutic dose (diclofenac 75-150 mg/day) for 3-7 days.

The method proposed for patients with severe exudative (acute) inflammation, verified clinical manifestations: purulent, profuse mucous discharge, the number of cells in microscopy at low magnification - more than 30 in the field of view in the samples urethral swab, urine, juice prostate cancer, sputum, bronchoalveolar fluid. In addition, there may be a large content of bacterial microflora at sowing detachable - the more than 10,000 microorganisms in men and 100,000 women in 1 ml.

To eliminate nonspecific bacterial flora offered ofloxacin. The choice of this antibiotic is made on the basis that hvostastym on the most frequent concomitant organisms with this type of inflammation: E. coli., enterobacteria, Enterococcus spp., Staphyloccus saprophiticus, at the same time, the drug in the proposed scheme does not affect the DNA isolation of mycoplasmas, that is not difficult to identify.

The rationale for the doses of the components of the treatment.

It is proposed to use the dose of fluoroquinolones from 100 to 250 mg 2 times a day for 3-5 days.

Doses less than 100 mg insufficient for eradication of bacterial pathogens, and a dose of more than 250 mg can potentially lead to the eradication of Mycoplasma antigen, removing Mycoplasma DNA or decline

Duration of use of ciprofloxacin or ofloxacin - 3-5 days, the application is less than 48 hours is not enough to erradicate gram-negative microorganisms [13], at the same time, the use of the drug 5 days or more prior to the sampling of the material for the diagnosis of mycoplasmal antigen can greatly reduce the amount, and consequently, adversely affect the detection of DNA of Mycoplasma.

To reduce the appearance of exudative inflammation, which makes difficult the separation of DNA of Mycoplasma, it is proposed to use non-steroidal anti-inflammatory drug. As a last choice fell on diclofenac, a drug with maximum anti-inflammatory effect of the means of this group, better portability when using comparable doses, with protivovospalitelnym [14, 15, 16]. To solve the task the application within 5 days can provide a pronounced anti-inflammatory effect. It is proposed to use the medium to high therapeutic doses - doses less than 1 mg/kg not able to provide the task for 2-3 days (approximately less than or equal to 75 mg/day), doses of more than 1.5 mg/kg can cause side effects (water retention) and, importantly, adverse interaction with fluoroquinolone drugs [12]. Therefore, clinical testing was conducted at doses of diclofenac 75-100 mg

The results of the experiment.

In the following table 1 presents the results of the diagnosis of mycoplasmosis according to polymerase chain reaction in patients with acute exudative inflammation before and after the proposed combination of anti-inflammatory therapy in patients with urogenital mycoplasmosis (verified by results of culture diagnostics on the specific environment to highlight with arginine-fermenting mycoplasmas)

Table 1
The patient's IDLeukocytes in smear BEFORE applying the proposed method to improve sample preparationThe results of the DNA diagnosis of Mycoplasmosis before applying the proposed method to improve about is podgotovki.* Leukocytes in smear after applying the proposed method to improve sample preparationThe results of the DNA diagnosis of mycoplasmosis when sampling at 5 days of combined anti-inflammatory and therapy
1.35-40+15-20+
2.55-60-20-30+
3.Cover the field of view-25-30-
4.35-45+20-25+
5..35-40+20-30+
6.40-50+25-30+
7.35-50+20-30+
8.Cover the field of view-25-30+
9.50-60-25-30+
10.35-40+20-30+
11.45-50-30-35-
12.50-60-25-30+
* + positive rez is ltate analysis on Mycoplasma

- negative test result for Mycoplasma

Thus, the original on the background of exudative inflammation in the urethra mycoplasmosis PCR diagnosis was detected in 6 of 12 patients. After the proposed method improve the preparation of the Mycoplasma was detected in 10 of 12 patients. That is, the proposed method improved sample preparation has improved the sensitivity of the method chain reaction (PCR) from 50 to 83%.

Comparison with previously known

Table 2 presents the results of the diagnosis of mycoplasmosis according to polymerase chain reaction in patients with acute exudative inflammation before and after the implementation of the method of mechanical removal urination and processing cotton swab with saline solution 0.9% NaCl in patients with urogenital mycoplasmosis (verified by results of culture diagnostics on the specific environment to highlight with arginine-fermenting mycoplasmas)

Table 2
The patient's IDThe original number of cells in the smear from the urethraThe results of the HRC-diagnosis of mycoplasmosisThe number of cells in the smear from the urethra after machiningThe results of PCR diagnostic micop is Osmose after machining
1.35-40+25-30+
2.50-60-25-30+
3.Cover the field of view-40-50-
4.35-45+20-30+
5.30-40+20-25+
6.45-50-35-40-
7.30-40+15-20+
8.Cover the field of view-40-50-
9.50-60-30-35+
10.30-40+20-25+
11.40-50-35-40-
12.50-60-30-35-

Effect: machining increases the detection of the pathogen additional 2 patients mycoplasmosis of 12 (from 41%to 58%, i.e. by 17%).

The result of the experiment using combined anti-inflammatory treatment of patients with severe exudative inflammation mo is eolovogo Department managed to find the cause of disease, another 4 people (from 50 to 83%). The proposed method improve the sample preparation can further increase the sensitivity of detection of DNA of Mycoplasma in patients with exudative inflammation by 33%, as compared to previously used methods of mechanical treatment by 16%.

The experiment was carried out in outpatient departments and PCR laboratory of the Institute of Microbiology of the Ministry of defense Kirov and Kirov state medical Academy, Department of dermatology and Department of therapy qualification development Department.

The program will increase the sensitivity of the PCR method to clarify the etiological diagnosis using the proposed method of sample preparation conducted in 60 patients with neonorange urethritis.

The proposed method improve the sample preparation has improved the sensitivity of PCR-based diagnostics in patients with severe exudative inflammation in an average of 33%.

LITERATURE.

1. Encyclopedia of clinical laboratory tests. Ed Nautica, editor-in-chief of the Russian edition Won. "Labelform", Moscow, 1997.

2. Rasin, S., Yogev d, Naot Y. Molecular biology and pathogenicity ofmycoplasmas. Environ Mol Biol Rev, 1998; 1094-1156.

3. Encyclopedia of clinical laboratory tests. Ed Nautica, editor-in-chief of the Russian edition Won. "Labelform", Moscow, 1997.

4. New genetic technologies. Collection 1. Interregional Association for the development of new genetic technologies. P.32.

5. Not rology. A guide for physicians in 2 T. Ed. Yestereve, Moscow, "Medicine", 1995, Vol.2. C.119.

6. Taylor-Robinson, D Infections due to species of mycoplasma and ureaplasma: An update. Clin Infect Dis 1996; 23: 671.

7. Balows. A., Hausler. WJ, Hermann. K.L., et al. (Eds.): Manual of Clinical Microbiology, 5 th ed. Washington, DC, American Society for Microbiology, 1991.

8. Clyde, W.A., Kenny, G.E., and Schachter, J.: Cumitech 19: Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections. Washington. DC, American Society for Microbiology, 1984.

9. Thomsen, AC, Undscov, HO. Diagnosis of mycoplasma hominis pyelonephritis by demonstration of antibodies in urine. J.Clin. Environ, 1979; 9:681.

10. Encyclopedia of clinical laboratory tests. Ed Nautica, editor-in-chief of the Russian edition Won. "Labelform", Moscow 1997. S (biomaterial and special requirements).

11. Encyclopedia of clinical laboratory tests. Ed Nautica, editor-in-chief of the Russian edition Won. "Labelform", Moscow 1997. S.

12. Encyclopedia of clinical laboratory tests. Ed Nautica, editor-in-chief of the Russian edition Won. "Labelform", Moscow 1997. 74.

13. Davia N. Gilbert, MD, Robert C. Moellering, Jr, MD, Merle A. Sande, MD. The Sanford Guide to Antimicrobial Therapy, Thirty - first Edition, 2001, p 23.

Method of increasing the sensitivity of PCR diagnosis of mycoplasmal infections in patients with acute exudative inflammation, characterized in that the patient in preparation for diagnosis injected antibiotic fluoroquinolone series - ciprofloxacin or ofloxacin dose of 100-250 mg 2 times a day, 3-5 days, and Nestea the command anti-inflammatory drug diclofenac at a dose of 75-150 mg/day, within 3-7 days.



 

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