Muzny, Christina A. MD*,†; Rivers, Charles A. PhD, MSPH*; Mena, Leandro A. MD, MPH†; Schwebke, Jane R. MD*
From the *Division of Infectious Diseases, University of Alabama at Birmingham, Birmingham, AL; and the †Division of Infectious Diseases, University of Mississippi Medical Center, Jackson, MS
The authors would like to thank Dr. Edward Hook, III, and Dr. Edwin Swiatlo for helpful discussions regarding preparation of this manuscript.
Correspondence: Christina Muzny, MD, Division of Infectious Diseases, University of Alabama at Birmingham, ZRB 242; 1530 3rd Avenue South, Birmingham, AL 35294. E-mail: email@example.com.
Received for publication October 24, 2011, and accepted February 7, 2012.
Trichomonas vaginalis (T. vaginalis) is the most common nonviral sexually transmitted infection in the world.1 The annual incidence of T. vaginalis infection among reproductive-age women (14–49 years) in the United States is estimated to be 3 to 5 million cases and the prevalence 3.1%.2
Although there is a wealth of data regarding prevalence of T. vaginalis infection among heterosexual US women,2–4 data are limited for other sexual risk groups, including women who have sex with women (WSW). Recent data from the 2006–2008 National Survey of Family Growth indicate that approximately 12.5% of American women aged 15 to 44 years engage in sexual activities with another woman at some point during their lifetime.5 The frequency and the mechanism of T. vaginalis transmission between women are not well understood, although sexual practices that provide a means for transfer of infected vaginal secretions may provide a plausible explanation (i.e., vaginal sex using hands, fingers, or penetrative sex toys).6 To date, this concept is best supported by a report of a metronidazole-resistant T. vaginalis infection found in 2 otherwise monogamous women who engaged in mutual masturbation.7 However, there are no data regarding the genetic relatedness of T. vaginalis isolates among mutually infected women involved in active sexual partnerships to determine whether their isolates are concordant. The objective of this study was to evaluate the use of the random amplified polymorphic DNA (RAPD) technique to delineate the genetic relatedness of T. vaginalis isolates among mutually infected WSW involved in active sexual partnerships and, in doing so, provide additional evidence regarding female-to-female transmission of T. vaginalis.
WSW participating in a study of sexually transmitted infection prevalence8 at the Mississippi State Department of Health (MSDH) STD Clinic in Jackson, MS, from February 2009 to October 2010, were selected for this study if they were in an active sexual partnership with another woman in the study at the time of enrollment and if both women were infected with T. vaginalis. A detailed sexual history of recent female (and male, if applicable) sexual partners during the past 3 months was obtained from all participants. For the purposes of this study, “sex” was defined to include vaginal, oral, or anal sexual contact. All women infected with T. vaginalis were treated according to the 2006 Centers for Disease Control and Prevention STD Treatment Guidelines.9
T. vaginalis was isolated from the vaginal vault of study participants during a standardized physical examination by culture in T. vaginalis InPouch media (Biomed Diagnostics, White City, OR). Axenic cultures were subsequently prepared in modified Diamond's medium by subculture to remove contaminating human cellular material, yeast, and bacteria. T. vaginalis DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). The concentration of DNA was determined by threefold serial dilution against a standard curve analysis of CT-values in a SYBR Green I real-time polymerase chain reaction assay for the T. vaginalis β-tubulin gene. Template DNA was diluted to a concentration of 5 ng/μl. RAPD was performed using 6 low-stringency polymerase chain reactions with 20 ng of template DNA (Illustra Ready-To-Go RAPD Analysis Kit, GE Healthcare Biosciences, Piscataway, NJ); this kit contained 6 random primers and appropriate reagents used in our methods. Reaction parameters were 95°C × 5 minutes/(95°C × 1 minute; 36°C × 1 minute; 72°C × 2 minutes) × 45 cycles/4°C hold. Amplicons were eletrophoresed in 2% agarose with tris acetate EDTA buffer prestained with ethidium bromide. Amplicon sizes were determined by comparison with a molecular weight ladder using Quantity One software (Bio-Rad Life Sciences, Hercules, CA). Hierarchical cluster analysis (Ward) of amplicon patterns was performed in JMPv9 (SAS, Cary, NC). Detailed sexual behavior data were reviewed if T. vaginalis isolates within a partnership were found to be concordant, focusing on sexual practices that may have provided a means for transmission of the organism. This study was approved by the Institutional Review Boards of the University of Mississippi Medical Center and the University of Alabama at Birmingham.
One hundred ninety-six WSW (W01-W196) were enrolled in the sexually transmitted infection prevalence study, of whom 35 (17.9%) were culture positive for T. vaginalis. Of these 35 women, 14 were actively involved in sexual partnerships with another woman in the study at the time of enrollment. Among these 14 women, 6 were involved in sexual partnerships with another woman enrolled in the study who was also infected with T. vaginalis. Thus, there were a total of 3 pairs of women who were mutually infected with T. vaginalis: W14/W19, W29/W32, and W46/W47. Of note, participant W14 returned to the clinic approximately 8 months after her enrollment visit and was again found to be culture positive for T. vaginalis. The isolates from her enrollment (March 2009: 03/09) and return visits (November 2009: 11/09) were also compared during this analysis. In total, 4 T. vaginalis isolate pairs were characterized using the RAPD method.
The RAPD technique generated a total of 31 bands ranging from 250 to 2061-bp across the 6 primers (Fig. 1). Six of the 31 (19.4%) RAPD bands were common among all isolates and not useful for discrimination. Primer 1 provided distinct patterns among 5 of the 7 clinical isolates (71.4%). Primers 2, 3, 4, 5, and 6 generated RAPD patterns that differentiated 71.4%, 57.1%, 71.4%, 85.7%, and 57.1% of the T. vaginalis isolates, respectively.
Of the 3 mutually infected pairs of WSW, 1 (W14/W19) shared a T. vaginalis isolate with the same RAPD banding patterns across all 6 primers (Fig. 2). W14 reported engaging in sexual activity with 2 partners (one female and one male partner) during the 3 months before enrollment in March 2009. She reported receiving oral sex from W19, a regular female partner, most recently 1 week before enrollment. She also reported having unprotected penile-vaginal sex with a regular male partner, most recently the day before enrollment. W19 reported only 1 female sexual partner (W14) during the same 3-month period. She denied recent sex with men, stating that her last male sexual encounter was in 2004. She reported giving and receiving oral sex from W14 and frequently sharing moist washcloths with W14 to cleanse their vaginal areas after oral sex. These washcloths were inconsistently washed with soap and water between uses.
Interestingly, the RAPD pattern of W14's T. vaginalis isolate from her return visit in November 2009 was discordant with the RAPD pattern of her isolate from March 2009, indicating that her initial treatment was successful, and that she had acquired a new T. vaginalis infection (Fig. 2). At her return visit, W14 reported that she had engaged in unprotected penile-vaginal sex with 2 male sexual partners since her initial study visit in March 2009, but had not had sex with W19 since April 2009.
W14 and W19 shared a T. vaginalis isolate with the same RAPD banding patterns, providing genetic evidence for indirect female-to-female sexual transmission of T. vaginalis, presumably through the use of washcloths contaminated with vaginal secretions, as there are no data to suggest that T. vaginalis is present in the oral cavity or transmitted during oral sex. The plausibility of this method of transmission is best supported by studies conducted in the late 1950s by Burch and colleagues, which demonstrated that T. vaginalis can survive on damp washcloths for periods of up to 24 hours.10 In addition, Adu-Sarkodie has reported probable transmission of T. vaginalis within a family in Ghana through the shared use of a bathing towel and sponge.11 Correspondingly, a recent cross-sectional study of adolescent girls aged 13 to 16 years in Zambia found that 24.7% (90/364) of women reporting to be virgins were infected with T. vaginalis, suggestive of nonsexual transmission. Among these women, a borderline association of T. vaginalis infection due to infrequent use of soap, OR 1.6 (95% CI: 0.9–2.7), and the type of toilet used (pit latrine/bush vs. flush), OR 1.6 (95% CI: 0.9–2.7), was present.12 Although it is generally accepted that T. vaginalis is transmitted almost exclusively by sexual intercourse,13 there have been additional reports of indirect methods of sexual transmission among WSW, including digital-vaginal transmission14 and transmission though mutual masturbation.7 In addition, iatrogenic transmission of T. vaginalis by a traditional healer to a patient following genital manipulation has also been recently reported from The Gambia.15 Despite these few instances, the data suggest that nonsexual transmission and indirect methods of sexual transmission of T. vaginalis are rare and occur infrequently.13,16–18 Nevertheless, clinicians should be mindful that these possibilities exist to counsel at-risk patients properly. Further studies are needed to improve our understanding of these potential methods of transmission of T. vaginalis and the frequency with which they occur.
Our study has several limitations. The majority of sexual behavior data collected was obtained by participant self-report and inherently limited by recollection bias or social desirability bias by the respondents. Thus, W14 and W19 could have participated in additional sexual activities that may have facilitated transmission of T. vaginalis (i.e., mutual masturbation, use of sex toys, etc.) that went unreported. In addition, although this study provides additional evidence that transmission of T. vaginalis can occur among WSW, it was not designed to determine the frequency with which this event occurs. A prospective analysis of WSW involved in sexual partnerships focusing on detailed sexual risk behaviors with female (and male) sexual partners and incidence of T. vaginalis infection over time is an important next step in this line of research. Finally, although RAPD is a powerful molecular technique that allows for investigation of genetic information without prior knowledge of the target organism's genome, there are several weaknesses inherent to this technique. Template DNA may be contaminated from other DNA sources (i.e., the host, other microbial organisms, etc.). In addition, if RAPD conditions are not optimized and consistent from test to test, results may not be reproducible. Resultant banding patterns may be difficult to interpret, not only in number and intensity, but also in position to a standard. Nevertheless, RAPD can be a valuable tool under controlled and well-characterized settings when no other genetic differentiation tools are available. It appears to be an informative assay for differentiating T. vaginalis isolates in this context.
The authors thank Dr. Edward Hook, III, and Dr. Edwin Swiatlo for helpful discussions in the preparation of this article.
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