Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples (original) (raw)
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Real-time PCR improve detection of Trichomonas vaginalis compared to conventional techniques
Comparative Clinical Pathology, 2012
Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. Clinical manifestations of symptomatic women are generally nonspecific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. The aim of the study was to compare a real-time polymerase chain reaction (PCR) assay with culture and wet-mount examination for the detection of T. vaginalis. This is a descriptive analytical study. Vaginal swabs from 504 female patients with suspected vaginitis were included in the study. They were subjected to culture, wet-mount microscopic examination (WM), and real-time PCR. Realtime fluorescence resonance energy transfer hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the b-tubulin genes. The result of the PCR was compared with culture and wet-mount microscopy. WM were positive for T. vaginalis in 60/504 cases (11.9%) and cultures were positive for T. vaginalis in 116/ 504 cases (23%). Real-time PCR was done on 50/504 specimens which was randomly chosen, 33/50 (66%) cases were positive for T. vaginalis (78% sensitivity), 25/50 (50%) of them were culture and wet-mount examination positive. Of them, 1/33 (3%) were culture positive and wet examination negative, 7/33 (21%) real-time positive were negative by culture while 17/50 (34%) real-time PCR negative cases were also negative by both techniques. The time taken for PCR assay was 4 to 8 h whereas positive protozoal cultures took up to 4 days. The Real-time PCR done in this study, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.
Cellular and molecular biology
Trichomoniasis is recognised as a major sexually transmitted disease (STD) in the world and may act as an acquired immunodeficiency syndromes (AIDS) co-factor by enhancing the transmission of human immunodeficiency virus (HIV). Diagnosis of Trichomonas vaginalis can be achieved by several methods, but sensitive detection means are stilllacking. ln this study a 2000-bp repeated DNA fragment of T. vaginalis was c1oned. Part of a conserved region of this insert was sequenced, two primers (TVK3 and TVK4) were chosen and a highly sensitive detection by polymerase chain reaction (PCR) was then developed for T. vaginalis. Ali strains ofT. vaginalis analysed with the se primers gave the expected 350-bp fragment and a 450-bp additional fragment. Sequence analysis of these PCR amplification products revealed that the 450-bp fragment contained the 350-bp with a 100-bp insertion characterised by a TGG microsatellite. A second primer set, namely TVK3 and TVK7 (determined at the border of the insertion), yielded PCR products of expected sizes. After amplification we were able to detect a single parasite. We also detected T. vaginalis in vaginal fluids of patients with STD. There was no reaction with human DNA or other infectious agents. lt appears that the two set primers are highly specifie of T. vaginalis and provide a useful tool for PCR diagnosis in asymptomatic and symptomatic patients especially among the HIV at risk individuals.
Sexually Transmitted Infections, 2007
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.
Introduction: Human trichomoniasis due to Trichomonas vaginalis is a curable sexually transmitted infection. It may lead to symptomatic vaginitis or asymptomatic carrier state. The symptoms and signs mimic other pathologies and conventional techniques of diagnosis have its own limitations. Therefore, the present study aimed to evaluate a newly established in-house PCR based assay on pfoB gene and compared it with conventional wet smear examination method and 18S rRNA gene based PCR technique for the diagnosis of T. vaginalis in symptomatic and asymptomatic subjects. Materials and Methods: Four hundred women in age group 20-57 years attending the Obstetrics and Gynecology out Patients Department (OPD) of Nehru Hospital attached to Post Graduate Institute of Medical education and Research (PGIMER) Chandigarh, India were included in the study. Based on the symptoms and signs, 344 (86%) women were categorized as symptomatic and 56 (14%) as asymptomatic. Vaginal swabs collected from all the women were processed by three techniques including wet smear, 18S rRNA and the pfoB gene based PCR techniques for the detection of T. vaginalis. Results: The main presenting symptom in majority of symptomatic patients was vaginal discharge. The highest numbers of T. vaginalis positive patients were found in the sexually active age group of 20 to 40 years. The amplifications of pfoB and 18S rRNA gene by PCR revealed significantly higher positive cases (20.7% and 18.6%, respectively) than the wet smear (6.6%) method. The diagnostic efficacy and kappa value estimated by the three techniques were 86.8-100% and 35.5-66%, respectively. Conclusions: The combined application of any two of the three techniques used in the present study may be useful for the diagnosis of T. vaginalis infection in symptomatic and asymptomatic subjects. This information may help clinicians to make a timely and accurate diagnosis.
Sexually Transmitted Infections, 2003
Objectives: DNA amplification techniques have become widely used for the diagnosis of sexually transmitted infections. For the detection of Trichomonas vaginalis, PCR techniques are not yet widely used despite the publication of several assays. The sensitivity and specificity of five independent primer sets were determined on self collected vaginal specimens obtained from female commercial sex workers. Methods: Self collected specimens were obtained from symptomatic and asymptomatic women attending a female sex workers clinic in Abidjan, Côte d'Ivoire. Two vaginal specimens were collected, the first one was processed for culture and the second was processed for PCR analysis. PCR techniques for trichomonads were performed, using the primers as reported by Riley (TVA5/TVA6), Kengne (TVK3/ TVK7), Madico (BTUB 9/BTUB 2), Shiao (IP1/IP2), and Mayta (TV1/TV2). An EIA amplicon detection method was designed for each of the primer sets. Results: True positive specimens were defined as culture positive and/or two positive PCR results with EIA amplicon detection in any combination. According to this definition a prevalence of 20% was obtained compared to 7% obtained by culture. The PCR primer set TVK3/TVK7 gave the highest sensitivity (89.2%). Poor sensitivities were obtained with the primer sets TV1/TV2 (60.2%) and TVA5/TVA6 (63.9%). PCR showed a sensitivity improvement of 2.4% up to 12% when EIA was used for amplicon detection. Conclusions: Overall, the sensitivities of the different PCR assays resulting from this study were lower than those previously described. These findings could be the result of the nature of the specimen population and suggests a strain variability.
Diagnosis of Trichomonas vaginalis infection by PCR
2007
Objective: To compare the sensitivity of PCR, wet preparation and culture in detecting Trichomonas vaginalis in urine and vaginal fluid. Methods: A PCR targeting the beta-tubulin genes of T. vaginalis was used for the detection of the organism in both vaginal swab and urine specimens from infected patients. Random urine samples were collected from 30 patients (23 females and 7 males), and tested for T. vaginalis by wet preparation and the Inpouch T. vaginalis culture systeme. Two vaginal swabs were collected by each woman. PCR detection. was carried out on samples negative by first methods. Results: The positive result was found in 28.57% in male urine and 39.13% in female urine samples, 65.21% in 1st swab and 78.26 % in 2nd swab by wet preparation. By culture, the male urine samples showed 42.85% positive, female urine 69.56% while 1st swab showed 86.95% positive and 2nd swab 91.30% positive. All negative cases by culture in urine and vaginal samples were tested by PCR, which showe...
18S ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis
Journal of clinical microbiology, 2000
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-posi...
European Journal of Obstetrics & Gynecology and Reproductive Biology, 2006
Objectives: The aim of this study was to compare wet mount-, Giemsa stain-, acridine orange fluorescent stain-, cultivation-and polymerase chain reaction (PCR)-based approaches to establish which method or combination of methods was most effective in the laboratory diagnosis of trichomoniasis. Study design: Out of 200 investigated patients with various gynecological complaints, Trichomonas vaginalis infection was detected in 27 (13.5%) by any of methods investigated. Among women with trichomonads, a typical clinical finding was presented in only nine. For analysis of sensitivity and specificity of the methods used, the receiver operating characteristic (ROC) curve concept with culture as a gold standard was applied. Results: Infection was diagnosed by wet mount in 14 (7.0%) women, by Giemsa stain in 11 (5.5%) and by acridine orange stain in 16 (8.0%) women. In 21 (10.5%) women, it was diagnosed by culture in Diamond's medium, and in 22 (11.0%) by PCR. For the initial diagnosis of trichomoniasis, wet preparation is the test that is widely available in most STD clinics, but its sensitivity is poor (66.67%). Giemsa stain shows a low sensitivity of 52.38%. Acridine orange shows reasonable sensitivity and specificity of 71.43% and 99.44%, respectively. The sensitivity and specificity of PCR (80.95% and 97.21%) did not exceed that of culture. Conclusion: With regard to the fact that trichomoniasis can have an atypical or even asymptomatic course, in order to accurately diagnose this disease, microbiological investigation is necessary. Comparison of different methods showed that at least two techniques, such as culture and acridine orange staining, have the potential for better diagnosis of T. vaginalis infection. PCR detection of infection has been demonstrated to be highly specific and sensitive, but its availability and cost effectiveness are in question. PCR could provide an alternative for laboratory diagnosis of trichomoniasis by culture. #
African Journal of Medicine and Medical Sciences, 2009
In the present study, three vaginal swabs were collected from 1469 females clinically suspected of having Trichomonas vaginalis (T. v) infection. All samples were screened by both wet mount and Diamond's culture media that was considered as the golden standard in this study. T. vaginalis gene detection by nested PCR using 4 primers targeting the Tv-E650 gene was performed on the preserved vaginal uncultivated samples corresponding to the culture positive vaginal specimens plus 30 randomly selected samples equaled to those obtained negative culture results. The prevalence of T. vaginalis infection among our patients was calculated according to the results of the golden standard culture method to be 1.43% (21 out of 1469). Wet preparation was positive for only 13 samples and missed 8 samples. PCR diagnosed 20 samples and missed one specimen that became positive after 4 days of cultivation. In this study, PCR for trichomonads does not appear to offer a diagnostic advantage and its sensitivity did not exceed that of culture. Successful culture of T. vaginalis requires only the multiplication of a single organism, the same as that needed for PCR. Therefore, the present work is highly recommending the use of Diamond's culture in the diagnosis of trichomoniasis in women.
TaqMan-Based Detection of Trichomonas vaginalis DNA from Female Genital Specimens
Journal of Clinical Microbiology, 2001
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, ΔRQ valu...