Trichomonas vaginalis Antimicrobial Drug Resistance in 6 US Cities, STD Surveillance Network, 2009–2010

Such isolates should undergo drug susceptibility testing periodically to detect emerging resistance.

T richomoniasis, caused by Trichomonas vaginalis, is one of the most common nonviral sexually transmitted diseases (STDs): annually, ≈248 million incident cases occur worldwide, and ≈7.4 million cases occur in the United States (1,2). The estimated US prevalence of T. vaginalis infection is 3.1%, with a higher prevalence among black women and women of low socioeconomic status than among other women (3). Trichomoniasis is a frequent cause of vaginitis and can contribute to premature rupture of membranes during pregnancy, preterm birth, low birth weight, and may facilitate HIV acquisition (4)(5)(6)(7).
The Centers for Disease Control and Prevention (CDC) (Atlanta, Georgia, USA) STD Treatment Guidelines recommends the use of a 5-nitroimidazole antimicrobial agent, either metronidazole or tinidazole, for the treatment of T. vaginalis infection (8). Metronidazole has been the mainstay of treatment for several decades; however, tinidazole has better in vitro activity and is well tolerated (8).
The reliance on a single drug class for treating T. vaginalis infections may be problematic if resistance to nitroimidazole becomes widespread in T. vaginalis strains. Three small studies that examined the prevalence of in vitro resistance in the United States have been conducted during the past 15 years, but they were limited in geographic scope (9)(10)(11). Our objective was to assess the prevalence of in vitro aerobic metronidazole and tinidazole resistance among a broad sample of T. vaginalis isolates from multiple geographic sites in the United States.

Methods
Demographic data and T. vaginalis isolates were collected from women attending 6 STD clinics participating in the STD Surveillance Network (SSuN). SSuN is a sentinel site surveillance network which, through the implementation of common protocols for collecting, reporting, and analyzing enhanced surveillance data, aims to improve the capacity of national, state, and local STD programs to detect, monitor, and respond rapidly to trends in STDs. T. vaginalis specimens from women undergoing Isolates were assayed for metronidazole and tinidazole susceptibility under aerobic conditions, according to the method developed by Meingassner and Thurne using serial dilutions of drug concentrations from 0.2 to 400 μg/ mL (12). The minimum lethal concentration (MLC) was the lowest dilution at which no motile trichomonads could be observed from an isolate assay. Isolates were tested in triplicate, and the assay was repeated twice. Control strains were CDC 085 (resistant) and CDC 520 (sensitive). If results differed, the modal result was used. Low-level resistance was defi ned as aerobic MLC 50-100 μg/mL, moderate-level resistance as 200 μg/mL, and high-level resistance as >400 μg/mL (13).
If multiple isolates were submitted from a single patient, we included the fi rst submitted isolate in the analytic dataset. We compared median MLCs of metronidazole and tinidazole by using the Wilcoxon matched pairs signed rank test to account for intra-isolate correlation. We assessed the prevalence of metronidazole and tinidazole resistance among isolates and compared the prevalence of resistance to each agent by geographic site by using the χ 2 test. We compared median metronidazole MLCs by geographic site by using the Wilcoxon rank sum test. Demographic and clinical data for women infected with a resistant isolate (metronidazole or tinidazole MLC >50 μg/mL) were compared with data for women infected with a susceptible isolate by using χ 2 or Fisher exact test for dichotomous data and t test for continuous data. p values were 2-tailed and considered signifi cant at p<0.05. Analyses were conducted by using SAS version 9.2 (SAS Institute, Cary, NC, USA).

Results
From April 6, 2009, through November 17, 2010, a total of 560 evaluable vaginal swab specimens were submitted from 538 women (range per woman, 1-3 swab specimens). Of these women, the median age was 28 years (range 13-67 years); 71% were African-American, 11% were non-Hispanic white, 11% were Hispanic or Latina, and 5% were of other race/ethnicity. Race/ethnicity data were missing for 2%. At least 1 previous episode of trichomoniasis was reported by 39% of women who submitted samples. Three percent of the women were pregnant, and none were HIV infected. Symptoms consistent with trichomoniasis (vaginal discharge, odor, or pruritus) were reported by 77% of women. By site, 80 (14.9%) women were from Birmingham, 94 (17.4%) were from Denver, 92 (17.1%) were from New York, 103 (19.1%) were from Philadelphia, 82 (15.2%) were from San Francisco, and 87 (16.2%) were from Seattle.
Sixteen women submitted 2 evaluable isolates, and 1 woman submitted 3 isolates. Among these 17 women, the median duration between sample collection was 128 days (range 2-392 days). Three women submitted the second specimens within 30 days of the initial submission (initial MLCs of metronidazole: 0.8-3.1 μg/mL), 3 within 60 days (initial MLCs 1.6-6.3 μg/mL), and 1 within 90 days (initial MLC 0.8 μg/mL). Data on sexual re-exposure were available for only 1 of these 7 women. For initial isolates from 2 women, MLCs of metronidazole were >12.5 μg/mL (50 μg/mL in both cases). In 1 case, the second isolate was collected 158 days after the fi rst, and the woman reported 12 sex partners in the preceding 3 months. In the other case, the second isolate was collected 308 days later, and the MLC of metronidazole for this strain was 0.8 μg/mL.

Discussion
To our knowledge, this study is the fi rst multisite evaluation conducted to assess the prevalence of in vitro T. vaginalis resistance in the United States. Although metronidazole has been used to treat T. vaginalis infections for ≈40 years, we found a low prevalence of in vitro metronidazole resistance. MLCs of tinidazole were lower than MLCs of metronidazole, and we did not detect tinidazole resistance.
The prevalence of in vitro metronidazole and tinidazole resistance is consistent with previously published US estimates. Three studies conducted in the southeastern United States among women attending STD or gynecology clinics from 1997 through 2005 found a metronidazoleresistance prevalence of 2.4%-9.5% (9)(10)(11). Most metronidazole-resistant isolates in these studies exhibited low-level resistance. In 2 of these studies tinidazole resistance also was tested: Krashin et al. did not detect tinidazole resistance (11), and Schwebke and Barrientes detected 1 isolate (0.6%) that exhibited low-level tinidazole resistance (MLC 50 μg/mL) among the 178 isolates tested (10). Among 91 isolates collected in Spain during 1995 and 1999, 2.2% exhibited low-level resistance to metronidazole (14). A small study conducted among women from Papua New Guinea found 21 (91%) of 23 studied isolates had MLCs of metronidazole of >50 μg/mL, including 4 (17%) with MLCs of 200 μg/mL (15). However, the sampling method used to enroll women was not described and may not have been systematic, thus substantially limiting the ability to estimate the population-level prevalence of resistance. Investigators in the United Kingdom reported that 1.7% of women treated for trichomoniasis during 1998-2002 appeared to have not responded to treatment and denied re-exposure; in vitro susceptibility data were not available (16).
Inconsistency does exist between in vitro susceptibility results and clinical outcomes of treatment, particularly for infections with low-level in vitro resistance. Clinical resistance and treatment failure have occurred with T. vaginalis isolates for which MLCs of nitroimidazoles were as low as 12.5 μg/mL, and treatment success has occurred in infections with T. vaginalis isolates for which MLCs of nitroimidazoles were 100-200 μg/mL (13). In general, however, elevated MLCs are associated with a greater likelihood of treatment failure. A recent evaluation of the utility of susceptibility testing in women for whom clinical treatment has failed found that treatment recommendations based on susceptibility results may have a benefi cial role in informing the clinical management of some women with persistent infection (17). T. vaginalis susceptibility testing is not available routinely; such testing should be conducted by a qualifi ed laboratory and is available at CDC (1-800-CDC-INFO).
As a cross-sectional evaluation of in vitro antimicrobial drug susceptibility, the study was not designed to detect clinical treatment failures. Multiple isolates were collected from 17 women. However, we did not systematically collect data on sexual re-exposure after treatment or adherence, so we were not able to determine whether any of these cases resulted from treatment failure. The MLCs of metronidazole for the initial isolates were low, suggesting that clinical resistance was unlikely. In 2 cases, the initial isolate exhibited low-level resistance (MLC of metronidazole 50 μg/mL) and a second isolate was later collected. Both of these cases were probably re-infections.
For isolates in our study, MLCs of tinidazole were lower than those of metronidazole, which supports the idea that tinidazole should be prescribed for patients whose infections do not respond clinically to metronidazole. This fi nding is consistent with results of previous studies which showed that tinidazole had better in vitro activity than metronidazole at similar molar concentrations (18). Tinidazole has a longer serum half-life than metronidazole . Susceptibility to metronidazole and tinidazole are defi ned as MLC <25 μg/mL, low-level resistance as MLC 50-100 μg/mL, moderate-level resistance as MLC 200 μg/mL, and highlevel resistance as MLC >400 μg/mL. and exhibits good tissue penetration (19), yet is more expensive than metronidazole. Although tinidazole and metronidazole are the only nitroimidazoles available in the United States, ornidazole, tenonitrazole, and nimorazole are available in Europe and could be alternatives to metronidazole. These agents are of the same drug class as metronidazole, however, and the emergence of clinically notable nitroimidazole resistance would be expected to adversely infl uence the treatment effectiveness of each of these agents.
This study had several limitations. First, the sample was limited to women attending STD clinics participating in SSuN; thus, our fi ndings are not representative of the general population. In addition, symptomatic women were likely to have been overrepresented because participating women were seeking care in STD clinics, and 2 of the sites sampled only symptomatic women. Also, although we believe this is the largest study of its kind, the sample size may not have been large enough to detect signifi cant differences across sites, nor to detect tinidazole resistance or high-level metronidazole resistance. That we did not detect isolates with such resistance suggests that its prevalence in this population is low. CDC occasionally receives isolates that are highly resistant to metronidazole or tinidazole, however (W.E. Secor, pers. comm.).
Although the prevalence of resistance is currently low in the United States, reliance on a single class of antimicrobial drugs heightens vulnerability if clinical T. vaginalis nitroimidazole resistance becomes widespread. Market forces alone are unlikely to spur the development of new anti-trichomonal drugs. Further evaluation of existing compounds and development of novel systemic treatment options are needed, and efforts to promote and support antimicrobial drug development and evaluation are warranted.
In summary, we found a 4% prevalence of low-level metronidazole resistance among T. vaginalis isolates from women attending several STD clinics throughout the United States. Periodic sentinel surveillance evaluations of T. vaginalis antimicrobial drug susceptibility should be carried out to monitor the possible emergence of resistance.