Shafir, Shira C. PhD*; Sorvillo, Frank J. PhD*†; Upcroft, Jacqui A. PhD‡; Upcroft, Peter PhD‡
From the *Department of Epidemiology, UCLA School of Public Health, Center for Health Sciences, Los Angeles, California; the †Data Collection and Analysis Unit, Los Angeles County Department of Health Sciences, Los Angeles, California; and the ‡Queensland Institute of Medical Research, The University of Queensland, Brisbane, Herston, Queensland, Australia
The authors gratefully acknowledge Dr. Lawrence Ash for assistance in experimental design and Dr. Patricia Johnson for supplying the T1 strain. The authors also acknowledge Dr. Roger Detels and Dr. Barbara Visscher for assistance in manuscript review.
Correspondence: Shira C. Shafir, PhD, UCLA School of Public Health, Department of Epidemiology, Center for Health Sciences Room 41-275, 650 Charles E. Young Drive, Los Angeles, CA 90024. E-mail: firstname.lastname@example.org.
Received for publication June 24, 2006, and accepted September 7, 2006.
Objectives: Two of the major diagnostic methods for Trichomonas vaginalis, wet mount and culture, rely on the continued viability of the organism. Methods to increase the viability of T. vaginalis in urine are needed.
Goal: The goal of this study was to develop a method that increases the time of viability of T. vaginalis in urine.
Study Design: Urine samples were inoculated with trichomonads, held at either room temperature or 37 °C, and processed through a column and frit, which was then placed in either a tube of culture medium containing antibiotics or a TV InPouch.
Results: The column and polyethylene frit system was found to increase the duration of viability for T. vaginalis from urine specimens at least 6-fold.
Conclusion: This novel method, which uses a column and frit system to increase the duration of viability of the organism, has the potential to increase the sensitivity of diagnostic tests.
TRICHOMONAS VAGINALIS IS THE MOST prevalent nonviral sexually transmitted infection worldwide.1 The disease can cause a wide variety of symptoms in women, but it is most often asymptomatic in men.2 The estimated global incidence of T. vaginalis infections is over 170 million cases per year with over 8 million new cases per year in the United States alone.3 In addition, high prevalence rates, averaging near 20% in women and 10% in men, have been observed across diverse populations in the United States. Moreover, infection with T. vaginalis has been shown to be a risk factor for transmission and infection with HIV as well as for adverse birth outcomes, including low birth weight and preterm delivery.4–7 Given the growing importance of T. vaginalis, information on the viability of this organism in diagnostic specimens is important. Two of the major diagnostic methods, wet mount and culture, rely on the continued viability of the organism, and survival of T. vaginalis, particularly in urine, is known to be poor.10 We report a method to increase the duration of viability of the organism, which may also serve to concentrate low parasite density specimens, particularly when a centrifuge is not available.
Materials and Methods
The strain of T. vaginalis used throughout this study, T1, originated from a clinical isolate from a patient in Taiwan and was maintained in modified Diamond's medium at 37°C in aerobic conditions.8,9 This particular strain is known to remain viable in urine for only 30 minutes.10
To assess viability of trichomonads in urine over time (and to attempt to increase the time between urine collection and test inoculation), 10 100-ml clean-catch urine samples were each inoculated with 100,000 trichomonads resulting in a final concentration of 1,000 trichomonads/mL in each specimen. After inoculation, the urine specimens were held at 37°C. For every 30 minutes over the next 5 hours, 5 mL was taken from each sample and processed through a disposable 25-mL column with a polyethylene frit (E&K Scientific, Campbell, CA) secured in place at the bottom. The frit was removed from the column and placed in a tube of Modified Diamond's medium including penicillin and streptomycin. At the same time points, another 5 mL was removed from each urine specimen and run through the column and frit. The frit was then placed into a TV InPouch (BioMed Diagnostics, White City, OR) for culture. The tubes and pouches were incubated at 37°C and examined daily for viable trichomonads. The experiment was repeated allowing the urine to sit at room temperature rather than incubating it at 37°C.
Ordinarily, urine is centrifuged once it is collected to increase the relative density of parasites in the specimen. However, this technique may not be practical in developing countries or in study situations in which the urine is collected and then transported to a distant laboratory. To attempt to collect T. vaginalis from low parasite density urine specimens without the use of a centrifuge, the same column and frit procedure was evaluated. A total of 100,000 parasites (strain T1) were dispensed from a stock culture, and 3 serial dilutions were conducted down to 100 parasites. Each dilution was added into 10 mL of clean-catch urine yielding final concentrations of 10,000 trichomonads/mL, 1,000 trichomonads/mL, 100 trichomonads/mL, and 10 trichomonads/mL, respectively. The contents of each tube were mixed and poured through the column and frit. The urine was drawn through the column through tubing fitted to the bottom of the column and a syringe. The frit was washed with 2 mL of Diamond's medium, then removed from the column and placed in a tube of medium containing antibiotics. Tubes were incubated at 37°C and examined daily using an inversion microscope for viable trichomonads. The experiment was repeated using the TV InPouch instead of culture tubes. The experiment was also repeated using different urine donors.
All specimens inoculated into a culture tube contained viable trichomonads for up to 4 hours after inoculation. Twenty percent of the specimens contained viable trichomonads 270 minutes after inoculation. No viable trichomonads were seen in any specimen that had been inoculated over 270 minutes previously (Table 1).
A slight difference was seen between the culture and InPouches. Viable trichomonads were present in all InPouch samples for up to 210 minutes after inoculation, but only 2 of the 10 specimens (20%) had viable trichomonads 240 minutes after inoculation. No viable trichomonads remained more than 240 minutes after inoculation (Table 2).
When urine specimens were held at room temperature, processed through the column and frit, and placed in culture tubes, viable trichomonads were seen in all specimens for 3 hours after inoculation. One of the 10 specimens had viable trichomonads for 210 minutes after inoculation, but no specimens contained viable trichomonads for more than 210 minutes.
When specimens were held at room temperature before being run through the frit and then inoculated into a TV InPouch, all specimens contained viable trichomonads for up to 3 hours after addition of the parasite to the urine. No specimens contained viable T. vaginalis after that point.
At higher inocula (10,000 and 1,000 trichmonads/mL), the culture tubes and InPouches were positive within 24 hours. All dilutions exhibited viable trichomonads within 48 hours of inoculation. No difference was seen between tubes of media and TV InPouches. However, by 72 hours, the bacterial overgrowth in the culture tubes, despite the addition of antibiotics to the media, was significant, whereas there was no such problem in the TV InPouch. Conversely, the InPouches were more difficult to read when the inoculum was low.
Previous studies have documented the reduced viability and/or recovery of T. vaginalis in urine specimens. As opposed to blood, which maintains a very narrow pH range, the normal values for urine pH range from 4.6 to 8.0, and alkalinity of urine has already been shown to retard the growth of T. vaginalis.11 Salt content of the urine could also have had an impact given that salt content exerts a significant and rapid lethal effect on T. vaginalis.12 Additionally, any other substances such as medicines or the presence of other sexually transmitted organisms might reduce the viability of the parasite. Further experiments need to be undertaken to identify the component of urine that results in loss of viability, which would allow for the development of techniques or substances to prolong the viability. Additional study of T. vaginalis viability using clinical isolates would also be of value.
By trapping the parasite in the filter while allowing flowthrough of the urine and washout with nutritive media, use of a column and frit system appears to either increase the duration of viability for T. vaginalis from urine specimens 8-fold, from 30 minutes to 4 hours if the specimen is held at 37°C and from 30 minutes to 3 hours when held at room temperature, or increase the likelihood of recovering viable organisms. Furthermore, the frit can substitute for a centrifuge in low parasite density urine specimens. Both functions may be particularly useful in developing countries where a centrifuge may not be available or where specimens are being collected a distance from where they will be tested.
Given the growing importance of T. vaginalis, efforts to increase the sensitivity of diagnosis are critical to both control efforts and epidemiologic studies. Current estimates of T. vaginalis in men, both prevalence and incidence, likely represent underestimates not only as a result of the inherent poor sensitivity of some currently used tests, but also because of delays in specimen processing. Future studies should ensure that specimens are processed rapidly or use methods such as described here to prolong viability. Development of easy-to-use and inexpensive antigenic techniques might serve to overcome these problems.13
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