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PubMed2000

18S ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis.

Trichomonas vaginalis remains the most common sexually transmitted parasite in the world and is considered a major risk factor in the transmission of the human immunodeficiency virus. A PCR technique using primers targeting a specific region of the 18S rRNA gene of T. vaginalis was developed. The PCR test was standardized using 15 reference strains, giving a single product of 312 bp in all strains. No amplification was observed when DNA from related organisms or human DNA was used as a target. The test was evaluated on 372 vaginal swab specimens and 361 urine samples from women attending infertility and obstetric clinics at two separate hospitals in Lima, Peru. Compared to T. vaginalis culture, the overall sensitivity and specificity of PCR of vaginal swab samples was 100% and 98%, respectively. The PCR of urine samples was 100% sensitive and 99.7% specific compared to culture of vaginal swab, but the sensitivity drops to 83.3% when compared to PCR of vaginal swabs. All culture-positive samples were found to be positive by PCR in either urine or vaginal secretion. None of the PCR-negative samples were positive by culture. The origin of the amplification was confirmed by digestion of PCR products with HaeIII. This PCR assay, which is easy to perform and has a high sensitivity and specificity, should be useful for routine diagnosis of T. vaginalis infection.

Animals↗
PubMed2000

Molecular epidemiology of metronidazole resistance in a population of Trichomonas vaginalis clinical isolates.

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNA T. vaginalis virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance to T. vaginalis resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for T. vaginalis infections.

Animals↗
PubMed2001

TaqMan-based detection of Trichomonas vaginalis DNA from female genital specimens.

A double-labeled fluorescent probe was designed and evaluated for detecting Trichomonas vaginalis DNA in a 5' nuclease (TaqMan) assay. The T. vaginalis-specific probe contains a 5'-fluorescein (5'-FAM) and a 3'-rhodamine (TAMRA) derivative. Female genital secretions were collected on Amplicor (Roche Molecular, Indianapolis, Ind.) swabs and by a transport system used for Chlamydia trachomatis and/or Neisseria gonorrhoeae DNA detection by PCR. Five hundred fifty-two female genital specimens, of which 248 (45%) were vaginal specimens and 304 (55%) were introital, were tested for both T. vaginalis DNA and viable microorganisms using the 5' nuclease assay and broth culture, respectively. Of these, 304 of 552 (55%) were also evaluated by direct microscopic examination for the characteristic motile organism. After resolving discrepancies, the comparisons produced an analytical sensitivity and specificity for the TaqMan-based PCR assay of 97.8 and 97.4%, respectively. As a result, DeltaRQ values (differences in fluorescence due to probe hybridization and resulting 5'-FAM cleavage from the specific PCR product) of > or =2.0 and < or =1.5 were established for T. vaginalis-positive and -negative cutoffs, respectively. DeltaRQ values between 1.5 and 2.0 were considered indeterminate. Overall findings revealed a high level of agreement between PCR and culture for detecting T. vaginalis. Potential benefits of the 5' nuclease assay include a greater sensitivity compared to direct microscopic examination and the ease of testing large numbers of clinical specimens in a significantly shorter turnaround time compared to culture.

Animals↗
PubMed2002

Development and validation of a PCR-based enzyme-linked immunosorbent assay with urine for use in clinical research settings to detect Trichomonas vaginalis in women.

Trichomonas vaginalis infection is highly prevalent worldwide and is associated with poor birth outcomes and enhanced human immunodeficiency virus transmission. Traditional detection methods rely on microscopic examination of vaginal specimens (wet mount) and culture, which can be insensitive and time-consuming. More than 3,000 women attending two sexually transmitted disease clinics were enrolled in this cross-sectional study to evaluate urine-based PCR for detection of T. vaginalis using a combined reference standard of wet mount and culture from vaginal swab. The prevalence of trichomoniasis in the population was 16.7% (502 of 3,009 women) using the reference standard. PCR with urine combined with agarose gel-based detection was 66.9% sensitive and 98.3% specific compared to the reference standard. Detection of PCR products using an unlabeled enzyme-linked immunosorbent assay (ELISA) improved the sensitivity to 86.4%, but specificity fell to 86.1%. Using a digoxigenin-labeled ELISA for detection of amplified T. vaginalis DNA from urine, the sensitivity and specificity of the PCR improved to 90.8 and 93.4%, respectively, compared to wet mount or culture from vaginal swabs. For clinical research settings in which vaginal specimens are not available and culture conditions are not feasible, urine-based PCR-ELISA may be useful for the detection of trichomoniasis in women.

Adolescent↗
PubMed2002

Timing of inoculation of the pouch makes no difference in increased detection of Trichomonas vaginalis by the InPouch TV method.

The InPouch TV is a method which combines a wet preparation and a culture method to detect Trichomonas vaginalis. The top portion of the InPouch TV essentially functions as a slide to be examined under the microscope. If the initial examination is negative, the specimen is pushed down into the bottom pouch, which serves as a broth for cultivation. The issue of timing has not been specifically addressed for optimal processing. To assess the effect of timing on the inoculation of the bottom pouch, we conducted a study designed to determine which procedure had better sensitivity, that of delaying inoculation of the bottom pouch until the initial examination on the top pouch is performed (method A) or that of immediately inoculating the bottom pouch (method B). In addition, we compared the sensitivity of the InPouch TV to that of the traditional wet mount. Fifty of 498 specimens were positive. Methods A and B had identical results: 31 specimens were initially positive regardless of transit time, and incubation yielded another 19 positives. The wet preparation detected 36 positive specimens. The sensitivities of the methods were 100% for the InPouch TV (including examination on receipt and after incubation) and 72% for the traditional wet mount. In conclusion, the InPouch TV method is more sensitive than the traditional method and no detectable differences were observed with timing of the inoculation of the top or bottom pouch.

Animals↗
PubMed2002

Detection of Trichomonas vaginalis on modified Columbia agar in the routine laboratory.

Broth culture of Trichomonas vaginalis is considered the "gold standard" for the diagnosis of trichomoniasis. Two studies were carried out to evaluate modified Columbia agar (MCA) for the isolation of T. vaginalis from clinical samples. Study I compared isolation on MCA to that on liquid medium with 889 vaginal samples. Out of 63 samples positive for T. vaginalis (7.1% of total), MCA identified 62 (98.4%) and broth identified 58 (92.1%). In study II, trichomoniasis was diagnosed within the scope of a screening program for a total of 39,585 men and women by culture on MCA and direct microscopy. Culture on MCA detected 199 (98.5%) and Gram staining detected 163 (80.7%) of 202 positive specimens. Wet-mount preparations used for symptomatic patients identified 103 (92.8%) of 111 cases. Culture of T. vaginalis from clinical samples on MCA is highly sensitive and reliable, as well as timesaving, and therefore suitable for screening of symptomatic and asymptomatic individuals.

Agar↗
PubMed2003

Use of the Roche LightCycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males.

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the "gold standard" and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.

Animals↗
PubMed2004

Evaluation of Xenostrip-Tv, a rapid diagnostic test for Trichomonas vaginalis infection.

An immunochromatographic strip test, Xenostrip-Tv, was compared to wet mount and PCR for the diagnosis of Trichomonas vaginalis infection in women. Of 428 specimens tested, 54 (12.6%) were positive by an "expanded gold standard," defined as either a positive wet mount and PCR test with primers TVK3 and TVK7 and/or a positive PCR test confirmed by a second PCR assay with primers TVA5-1 and TVA6; 26 (6%) were positive by wet mount, and 36 (8.4%) were positive by Xenostrip-Tv test. Since the Xenostrip-Tv test is rapid and easy to perform and proved to be more sensitive than wet mount, it should be considered as an alternative to wet mount for point-of-care diagnosis of trichomoniasis, especially in settings where microscopy is impractical.

Animals↗
PubMed2006

Use of an adaptation of a commercially available PCR assay aimed at diagnosis of chlamydia and gonorrhea to detect Trichomonas vaginalis in urogenital specimens.

Trichomonas vaginalis PCR using reagents from a commercially available assay for Chlamydia trachomatis and Neisseria gonorrhoeae was evaluated for detection of infection in women and men attending a sexually transmitted disease clinic. Evaluations included three primer sets, endocervical swabs, vaginal swabs and urine, and various storage conditions. The TVK3/TVK7 primer set was optimal in our hands with sensitivities ranging from 69.5 to 96.8%. In all comparisons, T. vaginalis PCR performed better than routine diagnostics using microscopy for women and culture for men (P > 0.05). The assay performed well for all sample types tested, and vaginal swabs were stable for up to 7 days at ambient temperature. Using samples prepared for, and reagents from, the C. trachomatis-N. gonorrhoeae PCR assay allowed incorporation of T. vaginalis PCR diagnosis into routine clinical testing.

Animals↗
PubMed1976

Comparison of four techniques for the routine diagnosis of Trichomonas vaginalis infection.

Specimens from 495 patients attending Johannesburg hospitals and family planning clinics were examined for Trichomonas vaginalis by microscopy of Giemsa (GS), Papanicolaou (PAP), and acridine-orange (AO) stained smears, and by culture in Feinberg-Whittington medium. Culture, Pap and GS stained smears from vaginal swabs yielded fewer positives than AO stained smears. Although Pap-stained cytological smears gave the highest number of positives, in 30% of these cases the presence of T.vaginalis could not be confirmed by examination of vaginal swabs. Of the positive AO-stained smears, 93% were also positive by at least one other technique.

Acridines↗
PubMed1987

Detection of Trichomonas vaginalis antigen in women by enzyme immunoassay.

An enzyme immunoassay (EIA) was developed for the detection of Trichomonas vaginalis antigen in vaginal swabs. Four hundred and eighty two women attending a sexually transmitted disease (STD) clinic were tested; 44 (9.1%) were positive by culture, 32 (6.6%) were positive by wet film examination, and 54 (11.2%) were considered to be positive for trichomonal antigen by EIA. Taking culture as the reference method, the EIA had a sensitivity of 93.2% and a specificity of 97.5%. The predictive value of a positive test was 82% and that of a negative test was 99.3%.

Antibody Specificity↗
PubMed2004

Comparison of latex agglutination, wet preparation, and culture for the detection of Trichomonas vaginalis.

OBJECTIVES: To compare the performance of three diagnostic methods for Trichomonas vaginalis infection-latex agglutination, saline wet mount, and culture. METHODS: Vaginal swabs from 3807 women attending antenatal clinics were tested for the presence of T vaginalis by latex agglutination. All positives and the following two negatives were tested by wet preparation and culture. RESULTS: The prevalence of infection by latex agglutination was 5.4%. Using an expanded gold standard based on the wet mount and culture results, the sensitivity of the latex agglutination test was 98.8% (95% CI 95.9 to 99.9) and specificity was 92.1 (89.2 to 94.5). The kappa index for test agreement was 0.93 for latex and culture and 0.88 for latex and wet preparation. CONCLUSION: The latex agglutination test is a highly sensitive test for detecting T vaginalis infection. It is a simple rapid test and has the potential for use in screening and diagnostic settings.

Animals↗
PubMed2006

Comparison of a TaqMan-based real-time polymerase chain reaction with conventional tests for the detection of Trichomonas vaginalis.

OBJECTIVE: To compare a TaqMan-based real-time polymerase chain reaction (PCR) with conventional PCR, culture, and wet-mount microscopy for the diagnosis of trichomoniasis in women. METHODS: Vaginal swabs from 119 women were tested for Trichomonas vaginalis by wet mount and culture. Paired vaginal lavage and urine specimens were tested by conventional and real-time PCR. RESULTS: Using an expanded "gold standard", defined as a positive culture result using vaginal swabs and/or a positive PCR test using TVK3/7 primers, the overall prevalence of T vaginalis in the study population was 65.5% (78/119). The detection rate of T vaginalis was 65.5% (78/119) and 36.9% (44/119) by conventional PCR using vaginal washings and urine specimens, respectively; 68.9% (82/119) by real-time PCR using vaginal washings and 61.3% (73/119) by real-time PCR using urine specimens. The sensitivities of conventional PCR using vaginal washings and urine and real-time PCR using vaginal washings and urine, compared with the gold standard were 100%, 56.4%, 100% and 76.7%, and the specificities of these tests were 100%, 97.6%, 82.9% and 97%, respectively. CONCLUSIONS: The real-time PCR test proved to be significantly more sensitive than culture and wet-mount microscopy, although its specificity was slightly lower than these tests. In addition, it was more sensitive, rapid and less time consuming than conventional PCR for the detection of T vaginalis.

Animals↗
PubMed1979

Rapid staining technique for Trichomonas vaginalis. Preliminary report.

A simple, rapid, staining method for the identification of Trichomonas vaginalis has been tested on cultured trichomonads and specimens of vaginal discharge. Fifty-eight stained slides of vaginal discharge were examined and trichomonads were correctly identified in the 31 specimens from patients with confirmed vaginal trichomoniasis. No false-positive results were obtained. This staining procedure could prove a useful addition to wet-film and cultural methods.

Adolescent↗
PubMed1979

Metronidazole-resistant Trichomonas vaginalis.

A 36-year-old woman with symptomatic metronidazole-resistant trichomonal vaginitis for 10 years had a total of 22 courses of treatment with either metronidazole or tinidazole according to different schedules. The minimum trichomonicidal concentration of metronidazole for the strain of Trichomonas vaginalis isolated from the patient was 160 microgram/ml compared with 1.25-10 microgram/ml for other freshly isolated strains. The former strain also showed a definitely decreased sensitivity to ornidazole and tinidazole (80 microgram/ml). The mechanisms behind the appearance of resistance in this clinical isolate are at present unknown and require further study from the theoretical as well as the therapeutic viewpoint.

Adult↗
PubMed1980

Urine culture for the detection of Trichomonas vaginalis in men.

Urine samples were collected from 248 men, 21 of whom were known contacts of women infected with Trichomonas vaginalis. This organism was cultured from only three of the 21 specimens from patients in the contact group. The cultural technique was shown to be capable of reliably detecting small numbers of organisms under practical conditions; it appears, therefore, that most male contacts shed relatively few trichomonads and that the infective dose for women must be correspondingly small.

Culture Media↗