The Composition of a Selective Media for Trichomonas vaginalis Isolation / 감염

BACKGROUND: Modified Diamond medium (MDM) supplemented with 10% heat-inactivated horse serum, streptomycin, penicillin G, and mycostatin is commonly used for the isolation of Trichomonas vaginalis from vaginal swab. But, judging from our experience, the above usual MDM antibiotic composition was frequently contaminated with facultative anaerobes, and isolation rate of T. vaginalis was no more than 12% in 142 korean woman patients whose chief complaints were foul odored, increased vaginal discharge. This isolation rate is low in comparison with reports of another countries including U.S.A (about 15~30%) and could be attributed to the prevalence of antibiotic resistance in Korea. So, we exploited more selective antibiotic compositions in modified Diamond medium for pure isolation of T. vaginalis. METHODS: We used new self-devised anaerobic pack for sample maintenance and tested several antibacterial and antifungal agent combinations in modified Diamond medium supplemented with 5% human erythrocyte lysate and 5% heat-inactivated human serum in the place of 10% horse serum with the object of increased and pure isolation of T. vaginalis. Several drugs and chemicals were tested to fourteen wild strains isolated in a local clinic, in the hope of finding the agents that have no effect on T. vaginalis growth in high drug concentrations. Anaerobic jar was used for culture of T. vaginalis and cell count performed in the improved Neubauer's haemocytometer. RESULTS: Strains of T. vaginalis grew batter in modified Diamond medium supplemented with 5% human erythrocyte lysate and 5% heat-inactivated human serum (mean 1.166X106, about 5.83 fold) than 10% horse serum (mean 2.0X105 after 48 hours culture), and their growth rate in the former was more rapid than the latter in early grow phase. On the basis of this results, we examined selectivity of modified Diamond media supplemented with several antibacterial and antifungal combinations by a double blind test. Isolation rate in the conventional modified Diamond's medium (combination A; 10% horse serum, streptomycin 1,200 microgram/mL, penicillin G 1,500 unit/mL, mycostatin 37.5 microgram/mL, pH 6.2) was 9/73 (12.3%) while in modified Diamond medium supplemented with 5% human erythrocyte lysate and 5% heat-inactivated human serum, isolation rates in various drug combinations were as follows; Combination B (cefazolin 100 microgram/mL, streptomycin 1,200 microgram/mL, clindamycin 150 microgram/mL, pH 6.5), combination C (bacitracin 14.6 unit/mL, streptomycin 1,200 microgram/mL, clindamycin 150 microgram/mL pH 6.5) and combination D (vancomycin 100 microgram/mL, streptomycin 1,200 microgram/mL, clindamycin 150 microgram/mL pH 6.5) were all 11/73 (15.0%). Combination D allowed the least bacterial growth rate. CONCLUSION: We consider that a new modified Diamond medium supplemented with 5% human erythrocyte lysate, 5% heat-inactivated human serum and combination D might be provide the highest selection for Trichomonas vaginalis pure isolation from vaginal swabs.

Resistant Patterns of Clinical Isolates of Trichomonas vaginalis against Antiprotozoal Agents under A Newly Devised Anaerobic Box / 감염

BACKGROUND: We thought that nitroimidazoles including metronidazole had been overused empirically for treatment of trichomoniasis in Korea. But there were not any reports about in vitro-drug susceptibility and distribution of resistant strains of Trichomonas vaginalis up to date. Therefore, we made an experiment in order to observe the susceptibility of clinical isolates of T. vaginalis to a variety of antiprotozoal agents. METHODS: Twenty-six strains of T. vaginalis isolated from 217 patients afflicted with the increased vaginal discharge were tested by Meingassner's microtiter plate method in newly devised anaerobic box, in which anaerobic and microaerobic conditions were more easily manipulated. The agents used in this study for testing the minimal lethal concentration (MLC) to the clinical isolates were as follows; nitroimidazoles, doxycycline, Zinc sulfate and gentian violet as chemotherapeutic agents and povidone-iodine as vaginal cleansing agents were studied. RESULTS: In anaerobic culture, according to anaerobic resistance cut-point (minimal lethal concentration >3.1 microgram/mL) proposed by M ller etc., metronidazole (MTZ)-, tinidazole (TNZ)-and ornidazole (ONZ)-resistant strains were four (4/26, 15.4%), two (2/26, 7.7 %) and two (2/26, 7.7%) strains, respectively. Among these resistant strains, two strains (G7 and G16) were resistant to two drugs and one strain (G20) resistant to three drugs concomitantly. Their resistance range was narrow as 6.25~12.5 microgram/mL. MLC of clotrimazole was > or = 2,000 microgram/mL in all strains, econazole was as high as 62.5~250 microgram/mL and miconazole was also high as 62.5~> or = 2,000 microgram/mL. In microaerobic culture (O2 concentration 100 microgram/ mL (aerobic resistance cut-point proposed by M ller etc.). MLC of doxycycline ranged 62.5 to 250 microgram/mL both in microaerobic and anaerobic conditions. All strains of T. vaginalis growed well in 3,000 microgram/mL of povidone-iodine. 22 strains (84.6%) among 26 T. vaginalis strains showed MLCs of 3.5 mM~7.0 mM to zinc sulfate. Gentian violet showed 15.6~62.5 microgram/mL of MLC. CONCLUSION: In absolute anaerobic culture, 4 strains (15.4%) among 26 T. vaginalis strains were resistant to metronidazole. But these 4 strains were not resistant in microaerobic culture depending on Miler's aerobic resistance cut-point (>50~100 microgram/mL), the value decided in normal O2 pressure. Vaginal PO2 is 0~28mm Hg (median 1 mmHg) at healthy or trichomonas-infected women. Therefore, we think that his aerobic resistance cut-point value is hard to be available in microaerobic condition and microaerobic resistance guide-line is to be established newly.