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Abstract
Objectives Microscopy is an insensitive method for detection of Trichomonas vaginalis, but is widely used because it is both rapid and inexpensive. Diagnosis of trichomoniasis by microscopy requires that motile forms be identified in vaginal fluid samples. However, microscopy cannot always be performed immediately after sample collection. The objective of this study was to assess the impact of sample storage at room temperature on trichomonad motility.
Methods Vaginal swab samples from 77 women positive for T vaginalis infection were collected to determine the impact of storage on wet preparations (swabs in plastic tubes with saline) and wet mounts (samples placed onto a glass slide with a coverslip). Samples were read at 400× every 30 min for the first hour and then once per hour thereafter until there were no motile trichomonads observed.
Results For wet preparations, motility was 100% at 30 min, 99% at 60 min and decreased by 3%–15% each subsequent hour, with samples having a lower density of trichomonads losing motility more quickly. Trichomonad motility diminished more rapidly in wet mounts compared with wet preparations, with a 20% decrement in motility in 60 min.
Conclusions These data suggest that vaginal fluid samples for diagnosis of trichomoniasis should be stored in saline rather than on microscope slides until they are examined under the microscope and samples should be evaluated by microscopy within an hour of collection. These findings also suggest that clinical sites which cannot perform microscopy within 1 h of sample collection should consider the use of other diagnostic tests.
- TRICHOMONAS
- INFECTIOUS DISEASES
- TESTING
- WOMEN
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Introduction
Trichomonas vaginalis is a commonly occurring sexually transmitted infection with a worldwide incidence in 2005 of 248.5 million among men and women, with an estimated 54.9 million of these occurring in the Americas.1 Infection with T vaginalis is often asymptomatic.2 Previous studies have shown this sexually transmitted infection is associated with premature membrane rupture, preterm labour, low birth weight of infants, and an increased risk of HIV transmission and acquisition in both men and women.2–7 More than 700 estimated new cases of HIV infection among women living in the USA have been attributed to infection with T vaginalis each year at an estimated cost of $167 million.3 Due to the potential problems and cost associated with T vaginalis infection, screening and treatment are crucial to halting the spread of the infection and limit the number of adverse outcomes.
Molecular amplification based methods are currently the most sensitive diagnostic tests for T vaginalis (90%–100%), whereas wet preparation of vaginal fluid (37%–92%), culture (81%–88%) or rapid enzymatic tests (83.3–94.7)8–21 each have wide ranges in reported sensitivity. However, molecular methods are not widely available in developing countries primarily due to the lack of instrumentation and trained personnel. This results in clinical laboratories sending samples to specialty testing laboratories delaying testing and receipt of the results for several days. Currently, the microscopic evaluation of a wet preparation of vaginal fluid remains one of the most widely used methods for detecting this organism because it allows point of care diagnosis at low cost. The 2010 CDC Sexually Transmitted Disease Treatment Guidelines state that diagnosis of trichomoniasis by microscopy ‘has a sensitivity of only 60%-70% and requires immediate evaluation of the wet preparation slide for optimal results’.22 One study evaluated the duration of trichomonad motility in wet mount preparations and reported that 35% of 65 specimens positive for motile trichomonads had no motile forms identified in as little as 30 min after a sample was placed on a slide under a coverslip.23 However, in clinical practice there can be delays in performance of microscopic evaluations for trichomonads due to the lack of availability of a microscope or other competing priorities for the clinician in completing the patient visit. The objectives of this study were to determine the length of time T vaginalis remains viable on a swab in a tube containing saline, and the survival time of T vaginalis once the sample has been placed onto a glass slide with a coverslip.
Methods
This study was approved by the institutional review board of the University Of Pittsburgh. Because this study was performed on de-identified samples which were to be discarded after their initial evaluation, this research study did not require written informed consent. Vaginal specimens were collected from women being seen for clinical care at two clinical sites located in Pittsburgh Pennsylvania as part of their urogenital evaluations for sexually transmitted infection screening. The clinician collected vaginal specimens on a Dacron swab (Thermo-Fisher, Pittsburgh, Pennsylvania, USA) and placed it into a 5 ml polystyrene tube (Sarstedt INC, Newton, North Carolina, USA) containing approximately 0.5 ml of sterile saline (Nephron Pharmaceuticals Corp., Orlando, Florida, USA). Wet mount preparations were made and evaluated by clinicians for motile trichomonads under 100× and 400× magnification. After samples positive for motile trichomonads were identified by the clinician, the samples were de-identified and transported to the research laboratory within 30 min for additional evaluation. All samples were transported from the clinics to the laboratory in an insulated cooler to keep the samples as close to room temperature (25°C) as possible during transport. Upon receipt in the laboratory, time and date of collection were noted. Wet mounts were made immediately upon receipt in the laboratory by rubbing the immersed swab around the inside of the tube to dislodge any trichomonads that were adherent to the side of the tube, removing the swab from the tube and pressing it against a glass slide to expel the liquid. The swab was then placed back into the tube and the liquid on the slide was covered with a coverslip. The average number of trichomonads was determined by counting the number of motile trichomonads in a given field at 400× for five fields and was recorded for the time point. The specimens were sampled in this manner once every 60 min for 8 h or until motile trichomonads were no longer identified. If trichomonads were still detectable at the end of a workday the specimens were left at room temperature and re-evaluated the following day (up to 24 h after collection).
To determine how long T vaginalis remains motile once the specimen is on the slide with a coverslip, the first slide was evaluated every 30 min for the first hour and then once an hour thereafter for the first 8 h or until no motile trichomonads were identified. Samples positive for motile trichomonads after 8 h were examined again at 24 h for continued determination of viability. The Kruskal–Wallis test was used to evaluate differences in the median survival time of T vaginalis stratified by number of trichomonads per high power field.
Results
A total of 77 vaginal specimens positive for T vaginalis were collected and evaluated. The average number of motile trichomonads observed per 400× field ranged from less than 1 to 139 at the time of sample collection. The survival time, defined as the time interval during which no more motile trichomonads were identified, ranged from 0.5 to 50 h. The specimens with fewer than 10 motile trichomonads per 400× field had a significantly lower median survival time compared with those having 10–50 trichomonads or those with having greater than 50 (see table 1).
Median survival times and ranges of Trichomonas vaginalis
The proportion of specimens having motile trichomonads at time points ranging from 30 to 1440 min (24 h) is shown in figure 1. At 30 min, 100% of the specimens contained motile trichomonads, and at 120 min, 96% retained motile trichomonads. For each subsequent 60 min there was a 4%–15% decrease in the number of specimens with motile trichomonads (see figure 1).
Comparison of trichomonad positive wet preparations and wet mounts from 30 min to 24 h.
A subset of 46 specimens was used to evaluate how long motile trichomonads could be identified after the specimen had been placed on a slide with a coverslip. Overall, the time to loss of motility was considerably shorter for these specimens with a range of 0–360 min. Those specimens having fewer than 10 trichomonads per 400× field had a significantly lower median survival time than those with 10–50 trichomonads or those with greater than 50 (see table 1).
Wet mounts with motile trichomonads had survival times ranging from 0 to 360 min (figure 1). In as little as 30 min there is a 9% drop in the number of wet mounts containing viable T vaginalis. Every 30 min after there was an 11%–28% drop in the number of wet mounts with motile trichomonads. The density of trichomonads in the initial sample was positively associated with survival time.
Discussion
The data from the present study support the recommendation from the US CDC to perform microscopy for trichomonads immediately following vaginal fluid sample collection. The data from the present study further suggest that the integrity of the sample is maintained longer when the vaginal swab is held in saline than when the wet mount is prepared and the sample is held under a coverslip. These data are relevant to clinical practice because wet mount microscopy is still widely used to diagnose infection with T vaginalis.
A strength of this study is that the concentrations of motile trichomonads for each sample at the time of sample collection were estimated and the samples were serially evaluated until there were no more observed for each of the samples collected. This allowed the identification of motility loss stratified by density of infection. Not surprisingly, the decrease in wet mount sensitivity over time was greater for vaginal fluid samples having a lower density of pathogens present. One limitation of the study was the lack of available personnel to perform hourly readings throughout the night. Samples were evaluated every hour for the first 8 h and then again after 24 h. Therefore, the estimates of viability are imprecise for the samples which lost motility between 8 and 24 h. However, the lack of precision for these estimates does not alter the conclusions of the study.
Direct microscopy for diagnosis of trichomoniasis is known to be an insensitive test for detection of this sexually transmitted pathogen. Multiple studies have shown that methods such as PCR, transcription-mediated amplification and rapid antigen testing (reported sensitivities of 77%–100%) have a higher sensitivity than wet mount and/or culture (reported sensitivities of 37%–100%).8–21 ,24 ,25 However, even if culture is being used for detection of T vaginalis, a wet mount evaluation can improve the overall detection of this parasite. A recent study showed that when wet mount or cultures are used independently of each other 17%–19% of women positive for T vaginalis may have a missed diagnosis.24 While PCR methods may be more sensitive, it is not available in clinical settings, making it unavailable for point of care testing and therefore delaying treatment. Despite its limitations, the low cost, wide availability and rapid results of the direct microscopy procedure have ensured its continued wide use.
Kingston et al23 reported that a third of vaginal fluid samples had no motile forms identified on a slide with a coverslip after as little as 30 min. In that study, the density of infection (number of motile trichomonads per high powered field) in each sample was not defined. In the present study, only those samples having the lowest pathogen load (fewer than 10 trichomonads per high powered field) had such a profound loss of motility in 30 min (figure 1).
For providers who continue to rely on direct microscopy for detection of T vaginalis, there are two important lessons. First, clinicians should be instructed to place the swab specimen directly into a saline tube until microscopy can be performed, preferably within 30 min. Second, specimens which are placed directly onto a slide with coverslip must be evaluated immediately or the result should be considered unreliable.
Because of the low sensitivity of wet mounts16 ,18–21 ,24 ,25 there are several factors that should be considered before determining whether wet preparations are adequate for diagnosis of infection with T vaginalis in a patient population. If samples cannot be evaluated quickly, there is an increased likelihood that the wet mount will be identified as negative even though a sexually transmitted pathogen is present. Preparation of wet mounts should be delayed until microscopy can be performed, whenever feasible. Training of the person performing microscopy is also important since there is wide variability in the skills of many clinicians reading wet mounts by microscopy. In the present study, 6% of the samples had less than 1 trichomonad per field. If a reader did not properly scan the entire wet mount made from the sample it could also be identified as a false negative, even if the sample can be evaluated immediately. Finally, if there is any delay in evaluating the wet mount preparation there is a significant decay in test sensitivity. The decreased motility of trichomonads on the wet mounts in such a short time period is most likely due to evaporation of the saline, with the greatest degradation in test sensitivity occurring in those samples having the lowest parasite density.
Given the low sensitivity of direct microscopy, the use of more sensitive diagnostic testing for detection of T vaginalis should be considered for women at risk of sexually transmitted disease.
Key messages
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Once collected, vaginal fluid samples should be evaluated immediately under the microscope.
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If samples cannot be evaluated immediately they should be maintained in a saline tube until they can be evaluated and not on a glass slide with a coverslip.
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There was a positive association between survival time and parasite density.
Acknowledgments
We would like to thank Carol Priest, Abby Jett and Dawn G Salerno for collecting and saving all the Trichomonas vaginalis positive samples.
References
Footnotes
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Contributors KAS, LKR and SLH were responsible for designing the study and drafting the manuscript. LKR and KAS were responsible for transporting samples to the laboratory, as well as reading the wet mounts and wet preparations. LAM was responsible for the statistical analysis.
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Funding This study was supported by the Microbicide Trials Network laboratory which is funded by 5 UM1-AI 068633.
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Competing interests None.
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Ethics approval University of Pittsburgh.
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Provenance and peer review Commissioned; externally peer reviewed.