Activities Of Sodium Nitrite In Combination With Antifungal Agents Against Trichophyton Rubrum In Vitro

Objective: Trichophyton rubrum is one of the most common pathogenic dermatophytes, mainly affects the skin, nails. It may be a violation of human hair now and then. Many overseas studies have shown that in patients with superFICal fungal pathogen analysis, the isolation rate of trichophyton rubrum accounted for the first. In addition, Jin Xuezhu et al had taken an analysis on pathogenic fungi isolated from patients with superFICal mycoses in Jilin province during the period of 15 years, which showed that the isolation rate of trichophyton rubrum in tinea capitis increased gradually from 10.6% in 1980s to 76.9% in late 1990s. Not only caused by trichophyton rubrum skin infection, recent reports had caused deep tissue infections such as puruLent tinea, granuLoma, etc., its incidence increased year by year. As the course of persistent fungal disease caused by trichophyton rubrum, difficult to cure and easy to recur, become one of the problems of clinical research urgently.For patients with tinea pedis caused by trichophyton rubrum treatment, recommended"1+1 combination therapy"program, that is, a topical antifungal agents plus an oral antifungal combination therapy, can shorten the course of treatment, improve the efficacy and patient compliance, reduce relapse rate. Current clinical application of antifungal agents against trichophyton rubrum disease has a better effect, but most of these drugs are expensive, toxic side effects, and with the extensive application of these antifungal agents, resistant strains emerging, as create difficulties for clinical treatment. To reduce adverse drug reactions and drug-resistant strains, the clinical often used in combination. Sodium nitrite is commonly used as food additive. Scientific Committee on World Food Health published in 1992, human security standard sodium nitrite intake to 0~0.1mg/kg body weight, click here for standard use and consumption, will not cause harm to the human body. Rich source of sodium nitrite, low-cost, overseas research had confirmed that sodium nitrite in acidic conditions, has strong killing effect on a variety of dermatophytes and yeast, and clinical application of the topical agent in the treatment of tinea pedis and onychomycosis is effective. Researchers in our department ever applied MTT determination the effect of sodium nitrite on trichophyton rubrum, the results displayed that sodium nitrite inhibited trichophyton rubrum; in 1.25mmol/L~160mmol/L concentration range of pure sodium nitrite on the trichophyton rubrum no cytotoxic effect, which at 1.25 mmol/L~10mmol/L concentration range, with the increase of the concentration effect, the inhibition was increased; in 10 mmol/L~160mmol/L concentration range of the inhibition of trichophyton rubrum was no significant difference. Electron microscopy revealed that sodium nitrite affect hyphae ultrastructure, manifested mycelial morphological changes and trichophyton rubrum membrane and cell wall destruction. They shows a dose-time dependency. However, there is no report yet about antifungal activities of sodium nitrite in combination with antifungal agents against trichophyton rubrum in vitro.In the past few years, for dermatophytes in vitro susceptibility test methods and results of interpretation there is no uniform definition of the standard until 2008, the National Clinical and Laboratory Standards Institute (CLSI) proposed filamentous fungi liquid-based dilution antifungal susceptibility testing program (M38-A program), the second improvement program (M38-A2 program), which increased the dermatophytes related content. According to this document and other literature, we combined sodium nitrite and other five antifungal agents against 30 clinical isolates of trichophyton rubrum in vitro susceptibility testing with micro-dilution method. To explore the combination of its antifungal activity in vitro and the best joint concentration, thus guided rational use of antifungal drugs, effective prevented and treated of trichophyton rubrum disease caused by fungal infection to provide evidence. Method:1 Strains separation, culture and identification: All of the 30 strains were clinical isolates obtained from the patients with tinea cruris and onychomycosis from the second hospital of Hebei Medical University. According to the characteristics under the microscope, and the colony characteristics on sabouraudand and potato culture medium to identification.2 In vitro antifungal susceptibility assay2.1 Single antifungal agents susceptibility testing against fungal in vitro Add 100uL of the two fold dilutions of antifungal drugs into 96-multiwell microdilution plate the 1st to the 10th row each line, the 11th and the 12th is drug-free control, and add 200uL and 100uL RPMI1640 respectively. The inoculum suspension was diluted 50 times with RPMI1640, and then add this diluted inoculum suspension to each well of the 1th to 10th and the 12th row. The microdilution plates were incubated at 30℃for 4 to 7 days, and were read visually after 100% growth in the control wells.2.2 Sodium nitrite and antifungal agents in cross combination testingAdd the antifungal agents into 96-multiwell microdilution plate with micropipettor. 50uL of sodium nitrite series solution was added to the 1st to 10th well each line in order, from the high concentration to the low. Antifungal drugs series solution 50uL was added to the 1st to 10th well each row in order, from the high concentration to the low. More operation as well as the single susceptibility testing. The microdilution plates were incubated at 30℃for 4 to 7 days , and were read visually after 100% growth in the control wells.3 statistical analysis:Date analyzed by SPSS 16.0 software. The comparison of MIC values between antifungal agents by Kruskal-wallis test, and further comparition between each two groups by one-way ANOVA after rank transform; Wilcoxon rank test was used to compare the MIC changes after antifungal agents in combination with sodium nitrite; the different FIC values compared among antifungal agents in combination with sodium nitrite and different concentrations of each drugs by Kruskal-wallis H test, further comparison between each two groups by Nemenyi test. A two-tailed P value of < 0.05 was considered to be significant.Results:1 Distributions of sensitivity of clinical isolates:In 30 isolates, there are 5 resistant isolates, 20 dose dependent susceptible isolates and 5 susceptible iaolates to itraconazole. All of the 30 isolates are susceptible to terbinadine. In 30 isolates , there are 1 resistant isolates, 3 dose dependent susceptible isolates and 26 susceptible iaolates to fluconaole. There is no resistant isolates to two or more antifungal agents in our research.2 The MIC values of sodium nitrite and other antifungal agents against trichophyton rubrum in vitro:The geometric means of MIC value in sodium nitrite, itraconazole , terbinafine, fluconazole, vorionazole and caspofungin are 40mmol/L, 0.3299ug/mL, 0.0155ug/mL, 4.8121ug/mL, 0.0640ug/mL and 1.5157ug/mL, respectively. The comparison among six agents by Kruskal-wallis H test,χ2=160.138, P<0.01, the difference has statistical significance. Further comparition between each two drugs by one-way ANOVA after rank transform, all of the differences have statistical significant, P<0.05.3 The MIC values of sodium nitrite in combination with other antifungal agents against trichophyton rubrum in vitroThe geometric means of MIC value in itraconazole, terbinafine, fluconazole, vorionazole and caspofungin after in combination with sodium nitrite are 0.1403ug/mL, 0.0214ug/mL, 5.7890ug/mL, 0.0532ug/mL, 0.5236ug/mL, respectively. Compared to single susceptibility testing by Wilcoxon rank test, the MIC velues of itraconazole and caspofungin are decreased, P<0.05, the difference has statistical significance; The MIC velues of terbinafine and fluconazol are increased, P>0.05, the difference has no statistical significance; The MIC velue of vorionazole is decreased, P>0.05, the difference have no statistical significance.4 The result of sodium nitrite and antifungal agents in cross combination testing:4.1 The FIC index of sodium nitrite in combination with different concentrations of itraconazole in vitro:Different concentrations of sodium nitrite combined with 2ug/mL, 1ug/mL, 0.5ug/mL, 0.25ug/mL, 0.125ug/mL, 0.0625ug/mL, 0.0313ug/mL, 0.0156ug/mL itraconazole respectively shows that: the percentage of FIC<1 are as follows: 3.3%(1/30), 3.3%(1/30), 16.7%(5/30), 20%(6/30), 50%(15/30), 30%(9/30), 10%(3/30), 13.3%(4/30); the 1≤FIC<2 proportion are 0%(0/30), 13.3%(4/30), 16.7%(5/30), 53.3%(16/30), 23.3%(7/30), 33.3%(10/30), 50%(15/30), 46.7%(14/30), respectively; the FIC≥2 proportion are 96.7%(29/30), 83.3%(25/30), 66.7%(20/30), 26.7%(8/30), 26.7%(8/30), 36.7%(11/30), 40%(12/30), 40%(12/30). By the Kruskal-wallis H test,χ2=33.827, P<0.001, the difference has statistical significant. Further comparition between each two concentrations of itraconazole, there is no statistical significance between 0.125ug/mL and both 0.25ug/mL and 0.0625ug/mL groups, P>0.05. The differences have statistical significance between 0.125ug/mL and the rest concentrations, P<0.05.4.2 The FIC index of sodium nitrite in combination with different concentrations of terbinafine in vitro:Different concentrations of sodium nitrite combined with 0.04ug/mL, 0.02ug/mL, 0.01ug/mL, 0.005ug/mL, 0.0025ug/mL, 0.0013ug/mL, 0.0006ug/mL terbinafine respectively shows that: the percentage of FIC<1 are as follows: 0%(0/30), 3.3%(1/30), 10%(3/30), 20%(6/30), 13.3%(4/30), 13.3%(4/30), 13.3%(4/30); the 1≤FIC<2 proportion are 3.3%(1/30), 36.7%(11/30), 36.7%(11/30), 36.7%(11/30), 43.3%(13/30), 43.3%(13/30), 43.3%(13/30), respectively; the FIC≥2 proportion are 96.7%(29/30), 60%(18/30), 53.3%(16/30), 43.3%(13/30), 43.3%(13/30), 43.3%(13/30), 43.3%(13/30). By the Kruskal-wallis H test,χ2=4.299, P>0.05, the difference has no statistical significant.4.3 The FIC index of sodium nitrite in combination with different concentrations of fluconazole in vitro: Different concentrations of sodium nitrite combined with 16ug/mL, 8ug/mL, 4 ug/mL, 2ug/mL, 1ug/mL, 0.5ug/mL, 0.25ug/mL, 0.125ug/mL fluconazole respectively shows that: the percentage of FIC<1 are as follows: 3.3%(1/30), 10%(3/30), 10%(3/30), 10%(3/30), 10%(3/30), 10%(3/30), 13.3%(4/30), 13.3%(4/30); the 1≤FIC<2 proportion are 10%(3/30), 26.7%(8/30), 43.3%(13/30), 43.3%(13/30), 36.7%(11/30), 40%(12/30), 36.7%(11/30), 36.7%(11/30), respectively; the FIC≥2 proportion are 86.7%(26/30), 63.3%(19/30), 46.7%(14/30), 46.7%(14/30), 53.3%(16/30), 50%(15/30), 50%(15/30), 50%(15/30). By the Kruskal-wallis H test,χ2=14.234, P<0.05, the difference has statistical significant. Further comparition between each two concentrations of fluconazole, all of the differences have statistical significance between 16ug/mL and any other concentrations, P<0.05; The differences between the rest each two concentrations have no statistical significant, P>0.05.4.4 The FIC index of sodium nitrite in combination with different concentrations of vorionazole in vitro:Different concentrations of sodium nitrite combined with 0.5ug/mL, 0.25ug/mL, 0.125ug/mL, 0.0625ug/mL, 0.0313ug/mL, 0.0156ug/mL, 0.0078ug/mL, 0.0039ug/mL vorionazole respectively shows that: the percentage of FIC<1 are as follows: 0%(0/30), 3.3%(1/30), 3.3%(1/30), 16.7%(5/30), 26.7%(8/30), 23.3%(7/30), 3.3%(1/30), 3.3%(1/30); the 1≤FIC<2 proportion are 3.3%(1/30), 6.7%(2/30), 23.3%(7/30), 33.3%(10/30), 43.3%(13/30), 33.3%(10/30), 43.3%(13/30), 43.3%(13/30); the FIC≥2 proportion are 96.7%(29/30), 90%(27/30), 73.3%(22/30), 50%(15/30), 30%(9/30), 43.3%(13/30), 53.3%(16/30), 53.3%(16/30). By the Kruskal-wallis H test,χ2=30.760, P<0.001, the difference has statistical significant. Further comparition between each two concentrations of vorionazole, there is no statistical significance between 0.0313ug/mL and both 0.0625ug/mL and 0.0156ug/mL groups. Between 0.0313ug/mL and the rest concentrations, all of the differences have statistical significance, P<0.05.4.5 The FIC index of sodium nitrite in combination with different concentrations of caspofungin in vitro:Different concentrations of Sodium nitrite combined with 8ug/mL, 4ug/mL, 2ug/mL, 1ug/mL, 0.5ug/mL, 0.25ug/mL, 0.125ug/mL caspofungin respectively shows that: the percentage of FIC<1 are as follows: 10%(3/30), 20%(6/30), 26.7%(8/30), 30%(9/30), 73.3%(22/30), 30%(9/30), 6.7%(2/30); the 1≤FIC<2 proportion are 10%(3/30), 6.7%(2/30), 3.3%(1/30), 50%(15/30), 10%(3/30), 33.3%(10/30), 46.7%(14/30), respectively; the FIC≥2 proportion are 80%(24/30), 73.3%(22/30), 70%(21/30), 20%(6/30), 16.7%(5/30), 36.7%(11/30), 46.7%(14/30). By the Kruskal-wallis H test,χ2=51.176, P<0.001, the difference has statistical significant. Further comparition between each two concentrations of Caspofungin, all of the differences have statistical significance between 0.5ug/mL and any other concentrations, P<0.05.4.6 The comparison result of the antifungal effect between the five antifungal agents after conmined with sodium nitrite:The FIC idex of itraconazole, terbinafine, fluconazole,vorionazole and caspofungin in combination with sodium nitrite shows that: the percentage of FIC<1 are as follows: 63.3% (19/30), 6.7%(2/30), 20%(6/30), 50%(15/30), 70%(21/30); the 1≤FIC<2 proportion are 26.7%(8/30), 36.7%(11/30), 33.3%(10/30), 20%(6/30), 13.3% (4/30), respectively; the FIC≥2 proportion are 10%(3/30), 56.7%(17/30), 46.7%(14/30), 30%(9/30), 16.7%(5/30). By the Kruskal-wallis H test,χ2=36.630, P<0.01, the difference has statistical significant. Sodium nitrite conbined with caspofungin showed the best synergistic reaction, followed by itraconazole and vorionazole. Further comparition between each two drugs, the difference between itraconazole to terbinafine and fluconazole have statistical significance, P<0.01. The difference between vorionazole to terbinafine and fluconazole have statistical significance, P<0.01. The difference between caspofungin to terbinafine and fluconazole have statistical significance, P<0.01. There is no statistical significance among itraconazole, vorionazole and caspofungin. There is no statistical significance between Terbinafine and Fluconazole, P>0.05. Conclusions:According to the program of M38-A2 from the United States National Clinical and Laboratory Standards Institute (CLSI) and relevant literatures, we use sodium nitrite joint commonly used antifungal drug itraconazole, terbinafine, fluconazole, and new antifungal agent voriconazole, caspofungin to conduct sensitivity tests of Trichophyton rubrum. The conclusions as follows:1 The MIC value of sodium nitrite against the clinical isolates of trichophyton rubrum is high in the RPMI1640 medium, and high concentration of sodium nitrite has antifungal activities in vitro.2 The MIC values of itraconazole and caspofungin decreased after in combination with Sodium nitrite in vitro, they showed a good synergistic reaction.3 0.125ug/mL concentration of itraconazole conbined with sodium nitrite showed the best synergistic reaction.4 0.5ug/mL concentration of caspofungin conbined with sodium nitrite showed the best synergistic reaction.5 The MIC values of terbinafine and fluconazole increased after in combination with sodium nitrite in vitro, they showed mainly a antagonistic reaction.