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Original Study

The Antimicrobial Effect of Boric Acid on Trichomonas vaginalis

Brittingham, Andrew PhD*; Wilson, Wayne A. PhD

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doi: 10.1097/OLQ.0000000000000203
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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

Parasites

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.

Statistical Analysis

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.

RESULTS

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.

Figure 1
Figure 1:
The effect of boric acid on the growth ofT. vaginalis. T. vaginalis (G3 strain) cultures were established in TYM media with increasing concentrations of boric acid. The density of intact and viable organisms, at the indicated time points, was monitored using a hemocytometer. Boric acid at concentrations of 0.2% and higher resulted in statistically significant growth inhibition at the 24-hour time point and beyond (*P < 0.001). Values shown represent the mean ± SE of 3 separate experiments.

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).

TABLE 1
TABLE 1:
MLC of Boric Acid forT. vaginalis

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.

Figure 2
Figure 2:
The restoration ofT. vaginalis growth after the removal of boric acid. After 24 hours of growth in either control media or media containing 0.2% boric acid, cultures were washed and resuspended to a density of 5 × 104 parasites/mL in fresh media with or without boric acid (arrow). Cell density was calculated as in Figure 1. There was no statistically significant difference in growth between cultures grown without boric acid for the entire experiment (control) and those cultures that were switched from 0.2% boric acid to control media after 24 hours of growth. Values shown represent the mean ± SE of 3 separate experiments.

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).

Figure 3
Figure 3:
The effect ofT. vaginalis growth and the addition of boric acid on the acidification of parasite growth media. T. vaginalis cultures were established in the presence of increasing concentration of boric acid, and growth was monitored as in Figure 1. The pH of the culture media was determined immediately after parasite inoculation (initial pH) and after 24 hours (A) and 40 hours (B) of growth. Values shown represent the mean ± SE of 3 separate experiments.

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).

Figure 4
Figure 4:
The effect of pH on the trichomonacidal activity of acetic, boric, and lactic acid. Cultures ofT. vaginalis were established in TYM media containing equal molar (32 mM; 0.2% for boric acid) concentrations of acetic acid, boric acid, or lactic acid. Before the inoculation with T. vaginalis, the pH of the media was adjusted using either HCl or NaOH. Growth inhibition at 24 or 40 hours was calculated relative to control media at an identical pH. Values shown represent the mean ± SE of 3 separate experiments.

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.

DISCUSSION

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.

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