Trichomonas vaginalis is a protozoan parasite and the causative agent of trichomoniasis, the most common nonviral sexually transmitted infection worldwide, with nearly 200 million cases reported annually. Although infection in men is generally asymptomatic, infected women present with a wide spectrum of clinical disease, ranging from asymptomatic carriage to severe vaginitis.1
Currently, nitroimidazoles (metronidazole and tinidazole) are the only recommended drugs used for treating trichomoniasis. With metronidazole refractory cases being documented, the emergence of low level resistance, and instances of patient intolerance (hypersensitivity), there is clearly a need for newer therapeutic approaches.2–4 Although a new systemically delivered compound for the treatment of trichomoniasis is the ideal, successful treatment in cases of metronidazole resistance or hypersensitivity using intravaginal therapies has been reported.5 In addition to the intravaginal delivery of compounds with known antimicrobial activity, such as paromomycin and betadine, compounds targeted toward modification of the vaginal environment have also been used, including lactic acid and acetic acid.
Topically applied (douches, washes, or gels) acetic acid or lactic acid has been used clinically in the treatment of not only trichomoniasis but also bacterial vaginosis.6–8 Although there are some reports regarding a direct antimicrobial effect of these compounds on the agents of bacterial vaginosis, often these treatments are reported to be aimed at restoring the normal acidic pH of the vagina.6,7,9
Recently, 2 reports have highlighted the effectiveness of treating trichomoniasis using intravaginally administered boric acid, a compound used more widely in the treatment of fungal vaginitis caused by Candida albicans as well as other species of Candida.10–13 Boric acid is an inorganic acid used in a wide and diverse array of clinical and industrial applications. Although used topically as a bacteriostatic and fungistatic agent, its mechanism of action remains unclear. In addition, little information on the pharmacology of topically applied boric acid exists, although it is generally considered safe for short-term use, with mild adverse effects.14
With reports appearing in the literature documenting its clinical effectiveness in treating trichomoniasis and its inclusion as a “home remedy” for treating vaginitis of multiple etiologies, we initiated studies designed to examine the in vitro effect of boric acid on T. vaginalis. We report that in vitro, boric acid inhibits the growth of both recent clinical isolates and laboratory strains of T. vaginalis. In addition, unlike acetic and lactic acid, the trichomonacidal effect of boric acid is independent of the pH of the culture environment.
We believe that these studies support the continued use of boric acid as a supplement to nitroimidazole compounds in the treatment of trichomoniasis and lay the groundwork for future studies aimed at determining the mechanism by which boric acid exerts its effect on T. vaginalis.
MATERIALS AND METHODS
T. vaginalis strains G3 (ATCC PRA-98) and C-1:NIH (ATCC 30001) were obtained from the American Type Culture Collection (Manassas, VA). Clinical isolates of T. vaginalis (PC11 and PC15) were obtained from the Polk County Health Department STD Clinic (Des Moines, IA) in compliance with protocols approved by the institutional review board for research involving human subjects at Des Moines University. For routine maintenance, cultures were grown in trypticase-yeast extract-maltose (TYM) medium adapted from the modification of Hollander Medium by Beal et al.,15 as described in Nielsen et al.16 Media were sterilized by autoclave and frozen at −20°C. Before use, media were completed with heat inactivated (56°C, 30 minutes) horse serum (10% final concentration), penicillin-streptomycin solution (50 U/mL penicillin G and 50 μg/mL of streptomycin sulfate), ferrous ammonium sulfate (0.01 mg/mL final concentration), and vitamin B12 (cyanocobalamin, 8 μg/mL final concentration). Routine culture was carried out at 35°C in 25-cm2 tissue culture flasks containing 5 or 10 mL of complete medium.
Clinical isolates were initially cultured using the InPouch TV culture system (Biomed Diagnostics Inc, White City, OR). Cultures were maintained for 3 to 5 days until abundant growth was observed. Cultures were then transferred and maintained in TYM medium as previously described, with the addition of chloramphenicol at a final concentration of 160 μg/mL. To facilitate the development of axenic cultures, trophozoites were collected by centrifugation (1500 × g for 7 minutes at 4°C) and washed in fresh medium during each passage. After approximately 30 days of culture in the presence of chloramphenicol, cultures were established without the additional antibiotic and without washing to confirm the lack of bacterial contamination. Stocks of axenic cultures were established and stored under liquid nitrogen.
Growth Curves and Effect of pH
To generate growth curves, T. vaginalis cultures were established at a density of 5 × 104 parasites/mL. At a given time point, the concentration of viable and motile trichomonads was determined using a Neubauer hemocytometer. To determine the effect of parasite growth on the pH of culture media, immediately after parasite inoculation, as well as after 24 and 40 hours of growth, 5 mL of culture was collected and cells were removed via centrifugation (1500 × g for 7 minutes at 4°C). The pH of the medium, free of cells, was then determined.
For assays examining the effect of boric acid on parasite growth, a stock solution of boric acid dissolved in complete TYM medium was first prepared and filter sterilized using a 0.2-μm filtration unit. This stock solution was used to prepare culture media containing boric acid at the final concentration necessary. In some assays, parasites were exposed to either control conditions or boric acid for the first 24 hours of growth, then washed and resuspended in fresh medium, with or without boric acid, for an additional 40 hours.
To compare the effect of acetic, boric, and lactic acid on trichomonad growth, and the effect of pH on these compounds, TYM media were supplemented with equal molar concentration of the 3 acids. The pH of each medium was then adjusted using either 2 M NaOH or 2 M HCl, as appropriate, to achieve the desired initial pH.
Minimal Lethal Concentration Assay
The minimal lethal concentration (MLC) of boric acid for T. vaginalis was determined at 37°C under aerobic conditions using a modification of the method developed by Meingassner and Thune.17 Briefly, cultures were established in 96-well microtiter plates at a density of 5 × 104 parasites/mL in the presence of increasing concentration of drug. The MLC was the lowest concentration of drug at which no motile trichomonad could be observed after 48 hours of exposure. Isolates were tested in triplicate, and the assay was repeated 4 times.
Differences in trichomonad growth were assessed using 1-way analysis of variance and the Tukey post hoc test. All statistical analyses were performed using SigmaStat (Systat Software, Inc, Dam Jose, CA) with a P value less than 0.05 deemed as significant.
In Vitro Susceptibility of T. vaginalis to Boric Acid
To assess the in vitro susceptibility of T. vaginalis to boric acid, cultures of T. vaginalis strain G3 were established in TYM media containing increasing concentrations of boric acid (0–0.7%), with growth monitored over a 40-hour period. Boric acid concentrations greater than 0.1% had a significant impact on T. vaginalis growth and viability (Fig. 1), with boric acid concentrations of 0.4% or higher inhibiting all parasite growth beyond the first 16 hours of cultivation (data not shown). A boric acid concentration of 0.2% inhibited parasite growth by approximately 50% at both the 24- and 40-hour time points.
The antitrichomonal effect of boric acid on T. vaginalis was assessed using the MLC assay.17 Using this method, the MLC of boric acid for the G3 strain of T. vaginalis is reported as 0.3% (Table 1). An additional common laboratory strain of T. vaginalis (C1:NIH) and 2 recent clinical isolates (PC11 and PC15) also demonstrated similar levels of susceptibility to boric acid, with MLCs of 0.5% or 0.6% (Table 1).
Collectively, these data demonstrate that at low concentrations (0.2%), boric acid reduces the growth rate of T. vaginalis, whereas at higher concentrations (≥0.4%), boric acid is lethal to trichomonads.
Reversibility of the Effect of Boric Acid on T. vaginalis Growth
To determine if the growth inhibitory effect of sublethal concentrations of boric acid on T. vaginalis was due to a permanent alteration of an undefined parasite biochemical or cellular process, T. vaginalis was grown for 24 hours in the presence of 0.2% boric acid, then washed and resuspended in medium lacking any boric acid. After the removal of boric acid, 24-hour treated cultures demonstrated a pattern of growth that was not significantly different from untreated control cultures (Fig. 2). Cultures grown in 0.2% boric acid for 24 hours, then washed and resuspended in fresh medium containing 0.2% boric acid, demonstrated increased growth suppression, perhaps due to the accumulated exposure time to boric acid.
These data suggest that the mechanism by which sublethal concentrations of boric acid suppresses T. vaginalis growth is reversible and not the result of a “lethal hit” or permanent damage, as described for metronidazole.18
The Effect of Boric Acid on T. vaginalis Is Independent of pH
We next assessed whether the effect of boric acid on T. vaginalis growth was dependent on the pH of the culture environment. The addition of boric acid to our base medium (TYM) had only a modest effect on the initial pH of the culture environment, reducing it from a pH of 7.00 ± 0.03 with no boric acid to 6.75 ± 0.02 at the highest boric acid concentration tested (0.7%; Fig. 3).
T. vaginalis growth in control media resulted in media acidification to a level of 6.59 ± 0.01 at 24 hours of growth and 4.84 ± 0.05 at 40 hours. Boric acid demonstrated lethality at concentrations of 0.4% or greater at these time points, with a level of acidification observed that was less than encountered during normal parasite growth (24 hours: 6.62 ± 0.01, 40 hours: 6.62 ± 0.03; Fig. 3A and B).
A similar observation was made when using a sublethal concentration of 0.2% boric acid, with growth being inhibited at a pH higher than typically observed in control cultures (Fig. 3).
These data demonstrate that the microbicidal effect of boric acid on T. vaginalis is not due to acidification of the medium.
Unlike Boric Acid, the Effect of Acetic and Lactic Acid on Parasite Growth Is pH Dependent
In addition to boric acid, formulations of lactic acid and acetic acid have been used in the treatment of vaginal infections including trichomoniasis.6,8,19,20 To compare the effect of these 3 acids on T. vaginalis, growth in equal molar concentrations of each acid was assessed. In addition, the relative impact of environmental pH on the effectiveness of each of these acids was also determined.
Boric acid, at a sublethal concentration of 32 mM (0.2%), resulted in an approximate 50% reduction in cell density at both the 24- and 40-hour time points, consistent with what was observed in Figures 1 and 3. Furthermore, this percentage inhibition was consistently observed regardless of the initial pH of the medium (pH 5, 6, or 7; Fig. 4).
In contrast to boric acid, the ability of acetic and lactic acid to reduce or prevent the growth of T. vaginalis was dependent on the pH of the growth medium. Both lactic and acetic acids had their greatest effect on T. vaginalis growth at pH 5, and no effect at an initial medium pH of 7. At a pH of 6, acetic acid had a significant impact on growth at the 40 hours time point only (Fig. 4).
These data demonstrate that unlike boric acid, the antitrichomonal activity of lactic and acetic acid is influenced by the acidity of the growth environment.
Currently, nitroimidazoles are the only class of Food and Drug Administration–approved compounds for treating trichomoniasis.21 In cases of drug hypersensitivity or initial treatment failure, few options exist outside desensitization or treatment with higher doses and/or prolonged treatment regimens. Alternative intravaginal therapies have been reported, although no large-scale clinical trials exist to support their effectiveness.5
It is widely reported than women with trichomoniasis, as well as bacterial vaginosis, often present with an elevated vaginal pH, reflective of an alteration in the composition of the bacterial flora.22 Historically, a multitude of probiotic and intravaginal acidic preparations have been used in an attempt to restore the “normal” vaginal flora and vaginal acidity.6,7,19,20,23 Particularly in the case of bacterial vaginosis, formulations (gels, suppositories, and washes) containing either acetic or lactic acid have been used in these efforts. Although some reports suggest that these acidification strategies are as effective as conventional antimicrobial therapy, other studies report these approaches to be ineffective in treating bacterial vaginosis.6,7,19,20 The use of lactic acid or acetic acid formulations in treating trichomoniasis, alone or in combination with other therapies, is less well documented but has been reported to aid in the resolution of infections refractory to other treatments.8,10,24
Boric acid is a weak acid often applied topically as a bacteriostatic or fungistatic agent; however, its exact mechanism of action remains unknown.11 Otic solutions used in treating otitis media and otitis externa often contain boric acid concentrations of 2% or greater, whereas boric acid creams and ointment used for dermatological conditions may contain concentrations of boric acid as high as 10%.25 Several reports support the usefulness of boric acid in treating vulvovaginal candidiasis, typically in the form of a gelatin capsule containing 600 mg of boric acid powder.12,13 The mechanism of action by which boric acid exerts its antifungal activity is unclear but may be related to disruption of cell wall synthesis and fungal morphogenesis.26,27
Recently, the effective use of boric acid in the treatment of trichomoniasis has been reported. Aggarwal and Shier10 reported the successful treatment for 2 women having recalcitrant T. vaginalis infection with a combination therapy which included boric acid. These authors cite the use of boric acid capsules as a way to acidify the vagina, although no pH measurements are reported. Muzny et al.28 reported on the use of boric acid capsules in the successful treatment for a patient with severe nitroimidazole allergy. Both articles conclude that boric acid may be a relatively safe, inexpensive, and effective alternative therapy in the treatment of trichomoniasis.
Our work sought to provide a foundation for in vitro studies aimed at addressing the effectiveness and mechanisms of action by which boric acid exerts its effect on T. vaginalis. Our studies demonstrate that at concentrations as low as 0.4%, T. vaginalis growth was completely inhibited, and that boric acid sensitivity was consistently observed in both laboratory strains and recent clinical isolates. It is difficult to draw correlates between the concentrations we observed as being effective in vitro to concentrations that T. vaginalis may encounter during the treatment for women with boric acid gelatin capsules, as there are no data available on the solubility and distribution of boric acid administered intravaginally.
Several groups have reported that the ability of boric acid to inhibit the growth of C. albicans is independent of pH.13,26 We report a similar observation regarding its effect on T. vaginalis. First, the acidification of the growth medium by the addition of boric acid was minimal and far less than what is typically observed during normal parasite growth. Second, when the pH of the medium was adjusted to a pH of 5, 6, or 7 after the addition of boric acid, there was no significant impact on the ability of boric acid to inhibit T. vaginalis growth. It is important to note that with a pKa of 9.24, over this wide pH range (5–7), most of boric acid will be in the acid form rather than the borate form, more lipid soluble and presumably able to enter the cell. The same would be true at normal as well as an elevated vaginal pH.
The pKa of acetic acid and lactic acid (4.8 and 3.8, respectively) may account for their difference in toxicity toward T. vaginalis at different pH levels. At the lower pH of 5.0, more acetic acid will be in the membrane-permeable, undisassociated acid form and will be able to enter the cell. As pH rises, more will be in the acetate form, which cannot readily enter cells and thus exhibits less toxicity. The relationship between pH and lactic acid toxicity is more complex, as lactic acid has been reported to exert its microbicidal activity through mechanisms other than cytosolic acidification.29,30 However, like acetic acid, its toxicity toward T. vaginalis is greatest at the lowest pH tested.
Our results demonstrate that boric acid exhibits potent antimicrobial activity toward T. vaginalis over a wide range of physiological pH. These data support previous clinical observations suggesting the use of boric acid as an alternative or supplementary treatment of trichomoniasis. In addition to future in vitro studies aimed at addressing the mechanism of action by which boric acid exerts its effect, larger-scale clinical studies are needed to assess its clinical usefulness.
1. Petrin D, Delgaty K, Bhatt R, et al. Clinical and microbiological aspects of Trichomonas vaginalis
. Clin Microbiol Rev 1998; 11: 300–317.
2. Schwebke JR, Barrientes FJ. Prevalence of Trichomonas vaginalis
isolates with resistance to metronidazole and tinidazole. Antimicrob Agents Chemother 2006; 50: 4209–4210.
3. Kirkcaldy RD, Augostini P, Asbel LE, et al. Trichomonas vaginalis
antimicrobial drug resistance in 6 US cities, STD surveillance network, 2009–2010. Emerg Infect Dis 2012; 18: 939–943.
4. Helms DJ, Mosure DJ, Secor WE, et al. Management of Trichomonas vaginalis
in women with suspected metronidazole hypersensitivity. Am J Obstet Gynecol 2008; 198: 370.e1–370.e7.
5. Muzny CA, Schwebke JR. The clinical spectrum of Trichomonas vaginalis
infection and challenges to management. Sex Transm Infect 2013; 89: 423–425.
6. Boeke AJ, Dekker JH, van Eijk JT, et al. Effect of lactic acid suppositories compared with oral metronidazole and placebo in bacterial vaginosis: A randomised clinical trial. Genitourin Med 1993; 69: 388–392.
7. Holley RL, Richter HE, Varner RE, et al. A randomized, double-blind clinical trial of vaginal acidification versus placebo for the treatment of symptomatic bacterial vaginosis. Sex Transm Dis 2004; 31: 236–238.
8. Jones CP, Carter B, Thomas WL. The treatment of resistant or recurrent vaginal trichomoniasis with lactic acid jelly and lactic acid douches. Gynaecologia 1960; 149 (suppl): 128–138.
9. O’Hanlon DE, Moench TR, Cone RA. In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide. BMC Infect Dis 2011; 11: 200.
10. Aggarwal A, Shier RM. Recalcitrant Trichomonas vaginalis
infections successfully treated with vaginal acidification. J Obstet Gynaecol Can 2008; 30: 55–58.
11. Prutting SM, Cerveny JD. Boric acid vaginal suppositories: A brief review. Infect Dis Obstet Gynecol 1998; 6: 191–194.
12. Swate TE, Weed JC. Boric acid treatment of vulvovaginal candidiasis. Obstet Gynecol 1974; 43: 893–895.
13. Van Slyke KK, Michel VP, Rein MF. Treatment of vulvovaginal candidiasis with boric acid powder. Am J Obstet Gynecol 1981; 141: 145–148.
14. See AS, Salleh AB, Bakar FA, et al. Risk and health effect of boric acid. Am J Appl Sci 2010; 7: 620–627.
15. Beal C, Goldsmith R, Kotby M, et al. The plastic envelope method, a simplified technique for culture diagnosis of trichomoniasis. J Clin Microbiol 1992; 30: 2265–2268.
16. Nielsen TJ, Pradhan P, Brittingham A, et al. Glycogen accumulation and degradation by the trichomonads Trichomonas vaginalis
and Trichomonas tenax
. J Eukaryot Microbiol 2012; 59: 359–366.
17. Meingassner JG, Thurner J. Strain of Trichomonas vaginalis
resistant to metronidazole and other 5-nitroimidazoles. Antimicrob Agents Chemother 1979; 15: 254–257.
18. Narcisi EM, Secor WE. In vitro effect of tinidazole and furazolidone on metronidazole-resistant Trichomonas vaginalis
. Antimicrob Agents Chemother 1996; 40: 1121–1125.
19. Andersch B, Forssman L, Lincoln K, et al. Treatment of bacterial vaginosis with an acid cream: A comparison between the effect of lactate-gel and metronidazole. Gynecol Obstet Invest 1986; 21: 19–25.
20. Holst E, Brandberg A. Treatment of bacterial vaginosis in pregnancy with a lactate gel. Scand J Infect Dis 1990; 22: 625–626.
21. Workowski KA, Berman S. Centers for Disease Control and Prevention (CDC). Sexually transmitted diseases treatment guidelines, 2010. MMWR Recomm Rep. 2010; 59: 1–110.
22. Linhares IM, Summers PR, Larsen B, et al. Contemporary perspectives on vaginal pH and lactobacilli. Am J Obstet Gynecol 2011; 204: 120 e1–120.e5.
23. Falagas M, Betsi GI, Athanasiou S. Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infect 2007; 13: 657–664.
24. Wood S, Kennedy CM, Galask RP. Prolonged vaginal and oral metronidazole for refractory Trichomonas vaginalis
: A case report. J Reprod Med 2007; 52: 1057–1058.
25. Clinical Pharmacology [database online]. Tampa, FL: Gold Standard, Inc.; 2014. Available at: http://www.clinicalpharmacology.com
. Accessed April, 10, 2014.
26. De Seta F, Schmidt M, Vu B, Essmann M, Larsen B. Antifungal mechanisms supporting boric acid therapy of Candida
vaginitis. J Antimicrob Chemother 2009; 63: 325–336.
27. Schmidt M, Schaumberg JZ, Steen CM, et al. Boric acid disturbs cell wall synthesis in Saccharomyces cerevisiae
. Int J Microbiol 2010; 2010: 930465.
28. Muzny C, Barnes A, Mena L. Symptomatic Trichomonas vaginalis
infection in the setting of severe nitroimidazole allergy: Successful treatment with boric acid. Sex Health 2012; 9: 389–391.
29. Alakomi HL, Skytta E, Saarela M, et al. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 2000; 66: 2001–2005.
30. Atassi F, Servin AL. Individual and co-operative roles of lactic acid and hydrogen peroxide in the killing activity of enteric strain Lactobacillus johnsonii
NCC933 and vaginal strain Lactobacillus gasseri
KS120.1 against enteric, uropathogenic and vaginosis-associated pathogens. FEMS Microbiol Lett 2010; 304: 29–38.