Evaluation of microscopy and polymerase chain reaction for diagnosis of symptomatic and asymptomatic female trichomoniasis

Nashaat E Nassef; Amera F Afif; Ashraf A Basuni; Mohamed F Abo El-Nasr; Amany F Atia

doi:10.4103/1687-7942.139689

RESEARCH ARTICLE

Year : 2014 | Volume : 7 | Issue : 1 | Page : 37-46

Evaluation of microscopy and polymerase chain reaction for diagnosis of symptomatic and asymptomatic female trichomoniasis

Nashaat E Nassef¹, Amera F Afif¹, Ashraf A Basuni², Mohamed F Abo El-Nasr³, Amany F Atia¹
¹ Department of Medical Parasitology, Faculty of Medicine, Menoufiya, Egypt and Saudi Arabia
² Department of Clinical Biochemistry, National Liver Institute, Rabigh Faculty of Medicine, King Abdulaziz Universities, Menoufiya, Egypt and Saudi Arabia
³ Department of Obstetrics and Gynecology, Faculty of Medicine, Menoufiya, Egypt and Saudi Arabia

Date of Submission	03-Aug-2013
Date of Acceptance	15-Dec-2013
Date of Web Publication	25-Sep-2014

Correspondence Address:
Amera F Afif
MD, Department of Medical Parasitology, Faculty of Medicine, Menoufiya University, Yassein Abdel-Ghaffar St., Shebin El-Kom, 23154, Menoufia, Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1687-7942.139689

Abstract

Background
Trichomoniasis is a sexually transmitted disease (STD) caused by the protozoan Trichomonas vaginalis. This infection is the most prevalent nonviral STD and may be symptomatic or asymptomatic. Accurate diagnosis is therefore necessary for specific treatment to facilitate control of infection and to prevent complication.
Objectives
The aim of the study was to evaluate different techniques for diagnosing trichomoniasis in symptomatic and asymptomatic patients.
Patients and methods
The present study included 85 nonpregnant female patients aged 18-50 years. According to the presence of symptoms related to vaginitis (discharge, itching, dysuria, dyspareunia, lower abdominal pain, and backache), patients were divided into two groups (symptomatic and asymptomatic). Vaginal samples were examined by wet mount, Giemsa stain, acridine orange stain (AO), and culture on modified diamond media in addition to PCR.
Results
The detection rate of T. vaginalis infection was 27.1, 28.2, 30.6, 35.3, and 57.1% using Giemsa stain, wet mount, AO, culture, and PCR, respectively. Giemsa stain, wet mount, and AO stain had a sensitivity of 76.7, 80, and 86.7%, respectively, when compared with culture. PCR detected all patients among the symptomatic and asymptomatic groups when compared with the culture method. The detection rate of T. vaginalis infection in symptomatic and asymptomatic patients by culture and PCR was 38.1 versus 51.8% and 27.3 versus 36.4%, respectively. The infection was commonest in the age range of 18-36 years.
Conclusion
The high sensitivity and specificity of PCR reported in this study offers a useful rapid screening tool and provides an excellent alternative for definitive laboratory diagnosis of T. vaginalis, particularly in asymptomatic patients, thus reducing spread and transmission of the infection as well as minimizing the complication sequels.

Keywords: asymptomatic, clue cells, diagnostic methods, polymerase chain reaction, trichomoniasis

How to cite this article:
Nassef NE, Afif AF, Basuni AA, Abo El-Nasr MF, Atia AF. Evaluation of microscopy and polymerase chain reaction for diagnosis of symptomatic and asymptomatic female trichomoniasis. Parasitol United J 2014;7:37-46

How to cite this URL:
Nassef NE, Afif AF, Basuni AA, Abo El-Nasr MF, Atia AF. Evaluation of microscopy and polymerase chain reaction for diagnosis of symptomatic and asymptomatic female trichomoniasis. Parasitol United J [serial online] 2014 [cited 2023 Dec 3];7:37-46. Available from: http://www.new.puj.eg.net/text.asp?2014/7/1/37/139689

Introduction

Trichomonas vaginalis is the cause of the most common sexually transmitted disease (STD) in the world ^[1] . In other countries, it was reported that the most risky population for trichomoniasis are young adults, especially teenagers, pregnant women, and sexually active women ^[2]. The flagellated parasite can cause vaginitis, cervicitis, cervical dysplasia, increased risk for postoperative infection, pelvic inflammatory disease, HIV acquisition and transmission, infertility, as well as adverse pregnancy outcome such as preterm labor, prostatitis, and urethritis ^[3] . It is cytopathogenic to the vaginal cells and may be associated with other STDs including HIV ^[4] . In the general population, infection is found in 5-10% of women, with an incidence of more than 200 million cases worldwide ^[5]. A current report suggested an incidence of 6-15% in asymptomatic women undergoing annual conventional cervicovaginal papanicolaou (PAP) smears, and 50% in high-risk STDs clinics ^[6] .

The occasional asymptomatic nature of trichomonal infection could also contribute to a long duration of infection and continued spread in the population ^[7] . Reports revealed multiple interplays of parasite and host factors, which lead to different clinical presentations ranging from asymptomatic to acute and chronic symptomatic states and complications. Many of them focused on parasite factors revealing cell to cell adhesion, pore-forming proteins, cell-detaching factors, excretion of extracellular proteinases, hemolytic activity, and differences in parasite intrinsic virulence. Host factors include the role of lactobacilli, humoral and cytokine responses, local pH, hormonal components, and free radical generation ^[8],[9],[10] .

Iron is an essential nutrient for the survival of pathogenic protozoa, especially the amitochondriate ones such as Trichomonas spp. These protozoa require higher extracellular iron concentration for their functional energy metabolism system.

This extracellular iron is bound to transferrin to be transferred to all cells, and at the mucosal surfaces it is bound to lactoferrin, which is an extracellular glycoprotein of the host immune system found in various mucosal secretions, including vaginal fluids. It was shown that the extent of ferric iron chelation by lactoferrin determines the clinical outcome of infection. Iron proved to affect the growth, cytoadherence, and virulence of T. vaginalis in vitro ^[11] . In a recent report, Horváthová et al. ^[12] indicated that iron has broad critical effects on T. vaginalis transcriptome as it regulates the expression of a single gene, whereas the expression of other paralogous copies of the gene is iron independent.

Accurate diagnosis of T. vaginalis, whether symptomatic or asymptomatic, is affected by many variables including patient factors, clinician's experience, specimen sampling and processing, test interpretation as well as the skill and expertise of those performing microscopic assessments. In addition, sensitivity, specificity, cost, ease of use, and time consumption by the different methods of diagnosis of T. vaginalis infection should all be considered ^[13] . Several methods are used for the diagnosis of T. vaginalis infection. Microscopic examination of wet mount preparations is a rapid method for diagnosis but with low sensitivity and specificity of 100% ^{[14],[15],[16]} . Cultures are considered the most reliable and gold standard diagnostic method, with greater sensitivity that may reach up to more than 80% and a specificity of 100% ^[14],[17] . The previously mentioned methods necessitate regular viable organisms, which is not usually suitable where transportation of specimens may be delayed ^[18] . Because cultivation methods are relatively slow and wet mount preparation lacks sensitivity, other staining and molecular probe methods have been used with direct clinical specimens ^[19] . Giemsa staining of smears is an easy, cost-effective, and a highly sensitive method of diagnosis ^[1] . Acridine orange (AO) is a nonspecific fluorochrome dye, examined by fluorescent microscopy. The sensitivity of AO is 67-70% in detection of T. vaginalis ^[15],[20] . An immunochromatography-based test (OSOM Trich test) for detection of T. vaginalis antigens directly from the vaginal swabs of symptomatic patients recorded 12 of 14 PCR-positive samples and one false-positive patient. Sensitivity of culture, wet mount, and OSOM Trich was 92.9, 64.3, and 85.7%, respectively, whereas specificity was 100, 100, and 99.1%, respectively ^[21] .

The use of PCR method helps in the detection of nonviable organisms and is also able to detect cells and target sequences in clinical samples that have undergone fixation or partial degradation ^[14],[22] . In clinical studies, reports of PCR using vaginal swab specimens have varied with sensitivities of 85-100% ^[23],[24] . PCR using urine specimens proved more sensitive than culture for the diagnosis of trichomoniasis, and was recommended for screening among men with recurrent prostatitis and urethritis as a part of routine checking in public health units to control trichomoniasis ^[25] .

The present study was undertaken to evaluate the different diagnostic techniques for diagnosis of trichomoniasis in asymptomatic female patients as compared to symptomatic ones.

Patients and methods

Type of study

This was a case-control study. The study was carried out on 85 nonpregnant female patients attending the Gynecology Outpatient Clinic at Menoufiya University Hospitals during the period from April 2008 to December 2010.

Patients

The patients were 18-50 years of age with various gynecological complaints but with no recent history of any antimicrobial therapy. They were classified into two main groups. The symptomatic group that included 63 patients complaining of symptoms suggestive of vaginal discharge with or without offensive odor, pruritus, lower abdominal pain, backache, dyspareunia, and dysuria, and the asymptomatic group that included 22 patients with no complaints suggestive of vaginitis and those who came with other gynecologic complaints or for annual examination. Women were excluded if they were pregnant, had gynecologic malignancy and/or vaginal bleeding, or if they refused speculum examination. Women were also excluded if they had undergone gynecological surgery and had history of STD or pelvic inflammatory disease within 1 month of the presentation. Patient information was collected on a standardized medical record form. Each patient voluntarily completed a questionnaire before examination, which covered basic data such as name, age, parity, marital status, first day of last menstrual period, history of sexually transmitted infection, history of abnormal cervical smear, use of contraception, use of treatment, and complaints. Patient selection, history taking, physical examination, local genital inspection, insertion of vaginal speculum, and samples collection were conducted by a gynecologist after consent from the patients. Diagnosis included vulvar erythema, discharge (color and odor), vaginal wall erythema and cervicitis, cervical mucopurulent erythema, colpitis macularis or strawberry cervix, and punctuate hemorrhage of the cervix.

Study design

For each patient, three speculum-assisted vaginal swabs were taken. The specimens were collected from the posterior vaginal fornix using sterile rayon swabs. The first swab was placed in 1 ml sterile saline solution and then used for wet mount, Giemsa, and AO staining. The second swab was inoculated in a culture tube, and the third was placed in 1 ml sterile PBS and stored at −20°C until use for PCR.

Methodology

The wet mount ^[15] was examined at ×400 to detect the trophozoites of T. vaginalis, with their characteristic morphological features and jerky motility. Giemsa-stained smear preparations ^[26] were examined microscopically at ×1000. AO-stained smear ^[15] was scanned under a fluorescent microscope at ×400, using selective beam splitter of TS 510 nm, barrier filter of G 247 nm, additional filter of G 249 nm, and excitation filter for narrow-band excitation of 255 nm. The second swab was cultured using Diamond's Modified Medium (Diamonds broth) ^[27] . Tubes containing fluid medium were warmed up to 35-37°C either in an anaerobic incubator for ˜1-2 h or by holding the tube under the arm or in the hand before inoculation. Warmed media were inoculated and incubated anaerobically at 37°C for 7 days. Cultures were examined daily from the tube sediment at ×400 magnifications to detect the motile trophozoites. The third swab was defrosted at room temperature and used for the PCR. DNA extraction was performed using DNA extraction kit (Strategic Medical Sales, Inc., 24381 Aurora Road, Suite B1 Bedford Heights, Ohio 44146, USA) as described by the manufacturers. The PCR protocol used and primer sequences, BTUB9 5'-CAT TGA TAA CGA AGG TCT TTA CGA T-3' and BTUB2 5'-GCA TGT TGT GCC GGA CAT AAC CAT-3', were applied according to Radonjic et al. ^[15] . PCR products, including positive and negative controls, together with a molecular weight marker 100-1000 base pairs were run on 3% agarose gel, stained with ethidium bromide, visualized by an ultraviolet source, and photographed ^[28] .

Statistical analysis

Data were collected, tabulated, and statistically analyzed by IBM personal computer and statistical package for science program version 11 (SPSS Inc., Chicago, USA). The χ² was used to determine the significant difference in detection of trichomoniasis in the studied age groups; P value less than 0.05 was considered significant. Sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy were calculated.

Ethical consideration

The study proposal was accepted by the Faculty Ethical Committee in March 2008. All patients included in the study were informed of the study objectives and written signed consent was taken from each one of them.

Results

Distribution of symptoms among the symptomatic group

Vaginal discharge was the most common symptom (77.8%), followed by vulvar pruritis (60.3%) and dysuria (41.4%). Lower abdominal pain and backache (25.4%) and dyspareunia (19%) were less common symptoms [Figure 1].

Figure 1: Distribution of symptoms among the trichomoniasis symptomatic group.

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Detection rate of trichomoniasis among different age groups by culture technique

This was higher in ages ranging from 31-36 years (58.8%), followed by ages 25-30 years (45.5%) and 18-24 years (33.3%). It was lower in the age ranges 37-42 years (12.5%) and 43-50 years (11.1%). The difference between data in the studied groups was significant (P < 0.05) [Figure 2].

Figure 2: Detection of trichomoniasis among different age groups by culture technique (ƒÓ2 = 11 .09).

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Comparison of different methods for diagnosis of trichomoniasis in the symptomatic and asymptomatic groups

Culture and AO stain methods [Figure 3], [Figure 4], [Figure 5] gave the same percentage (27.3%) of positivity in the asymptomatic group as compared with 38.1 and 31.7%, respectively, in the symptomatic group [Table 1]. Both wet mount and Giemsa stain methods [Figure 3], [Figure 6] and [Figure 7] recorded 22.7% positivity in the asymptomatic group, and 30.2 and 28.6% [Table 1] in the symptomatic group. PCR [Figure 8] detected all patients among the asymptomatic and symptomatic groups when compared with the culture method. The detection rate of trichomoniasis in asymptomatic and symptomatic patients by culture and PCR was 27.3 versus 36.4%, and 38.1 versus 57.1%, respectively [Table 1].

Table 1: Comparison of different methods for diagnosis of trichomoniasis in the asymptomatic and symptomatic groups

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Figure 3: Wet mount smear from culture showing Trichomonas vaginalis trophozoites with nucleus in anterior portion and flagella (arrows) (×400).

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Figure 4: Giemsa stain showing pear-shaped T. vaginalis trophozoite with dark nucleus (arrow b) and flagella (arrow a) (x1000).

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Figure 5: Giemsa stain showing clue cell (arrow a) and pear-shaped T. vaginalis trophozoites (arrow c) with dark nucleus (arrow b) and flagella (x400).

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Figure 6: Acridine orange fluorescent staining showing brick red T. vaginalis trophozoites (arrow b) with a yellow banana-shaped nucleus (arrow a) (×400).

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Figure 7: Acridine orange fluorescent staining showing brick red T. vaginalis trophozoites (arrow b and c) with a yellow banana-shaped nucleus (arrow a ) (×400).

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Figure 8: Agarose gel electrophoresis of PCR product. Lane 1: DNA ladder, lane 2– 5 and 7: positive results, lane 6: negative result.

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Correlation between culture and polymerase chain reaction in symptomatic and asymptomatic patients

PCR revealed positive infection in a total of 44 (51.8%) patients; eight (36.4%) were asymptomatic and 36 (57.1%) were symptomatic. The PCR confirmed all 30 patients who were diagnosed by culture and an extra 14 patients who were missed by culture. Therefore, culture gave a sensitivity of 68.2% and specificity of 100% compared with the PCR technique [Table 2].

Table 2: Correlation between culture and PCR in symptomatic and asymptomatic trichomoniasis patients

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According to these results, sensitivity of wet mount, Giemsa stain, AO stain, and PCR proved to be 80, 76.7, 86.7, and 100%, respectively, when compared with the culture. In addition, the accuracy of each test was 92.9, 91.8, 95.3, and 83.5%, respectively [Table 3].

Table 3: Sensitivity, specifi city, positive predictive value, negative predictive value, and accuracy of diagnostic methods compared with culture in patients tested for trichomoniasis

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It is relevant to note in [Figure 7] that the Giemsa stained trichomonad is associated with a granular vaginal epithelial cell suggestive of a 'clue cell'. This type of cell is coated around the edge by coccobacilli of Gardnerella vaginalis, which causes bacterial vaginosis ^[29] , and is related to the decrease in numbers of predominant bacteria in the vagina and consequent change in the normal acidic pH level of the vagina and the oxygen depleted environment ^[30] . This is in agreement with T. vaginalis, which is also anaerobic and grows best in oxygen-free low-acidity environments achieving maximum growth and metabolic function at a pH of 6.0 ^[31] .

Discussion

The high incidence of T. vaginalis infection worldwide and coinfection with other STDs render trichomoniasis a compelling public health concern. Notably, it was suggested that trichomoniasis may facilitate HIV transmission ^[32] and is associated with adverse pregnancy outcomes, infertility, postoperative infections, and cervical neoplasia ^[33],[34] . Accurate diagnosis is necessary for specific treatment that facilitates control of the infection and prevents complications. It is estimated that untreated T. vaginalis can persist for up to 5 years in women and is highly transmissible between partners ^[35] .

The objective of the present study was to evaluate the molecular techniques such as PCR for diagnosis of trichomoniasis compared with wet mount, staining, and culture techniques in the different age groups of asymptomatic and symptomatic women. As trichomoniasis is a STD, its prevalence is expected to reflect the period of highest level of sexual activity in a community. In our study, the detection of infection with respect to age was the highest in age range 31-36 years (58.8%), which may thus be considered the period of highest level of sexual activity for women in our community locally exposed to STD, followed by the levels in the age range 25-30 years (45.5%) and 18-24 years (33.3%). However, one report ^[36] indicated that trichomoniasis was most common between the ages of 16 and 35 years; another report ^[37] showed a high rate in women over 45 years of age. Another study ^[38] reported that cultures proved positive in 22.7% of women who were 18 years of age and younger as compared with 6.8% in those who were 64 years of age and older, the rate being the highest in the age group 36-45 years (43%).

In the present study, 77.8% of the symptomatic group reported discharge, 60.3% had pruritis, 41.4% had dysuria, 25.4% had lower abdominal pain and backache, and 19% had dyspareunia. Discharge and pruritis proved to be the most common symptoms. These results are in agreement with the study by Kassem and Majoud ^[39] who reported in their research that 93.1% of infected women suffered from vaginal discharge, 81.48% had burning, 79.39% had vulvar pruritus, 40.47% had dyspareunia, and 21.43% had dysuria. Akinloye et al. ^[36] also reported that 68% of patients infected with trichomoniasis had discharge (vaginal and urethral) and 42.1% had dysuria.

With respect to the infection rate detected by the culture method used among the symptomatic and asymptomatic groups in our study, 38.1% were positive in the former group as compared with 27.3% among the latter. Another research ^[40] reported 1.6% positivity in symptomatic patients as compared with 0.5% in asymptomatic ones, and concluded that T. vaginalis is associated with higher incidence rate in patients with symptomatic genitourinary disease. In contrast, 1.1% of asymptomatic patients were found infected with T. vaginalis in comparison with 0.8% among the symptomatic patients ^[41] . Dharma et al. ^[42] also revealed that 9.5% were asymptomatic compared with 28.5% of patients who were symptomatic. Of note, Smith et al. ^[3] reported that ˜50-80% of women infected with T. vaginalis are asymptomatic. Therefore, routine screening for the presence of infection in both symptomatic and asymptomatic patients is important.

Each laboratory method used in the diagnosis of trichomoniasis has its own advantages and faults. Direct microscopic examination by wet mount was the first to be implemented and remains the method most commonly used. Different reports indicated that the sensitivity of wet mount is between 35 and 80% when compared with the culture method [15,43-46]. In our study, wet mount showed 80% positivity and 100% sensitivity in a total of 30 positive patients detected by culture method, constituting 83.3% asymptomatic patients and 79.2% symptomatic patients. The reliability of wet microscopy in the diagnosis of T. vaginalis as compared with culture was confirmed by Ojuromi et al. ^[47] who recorded 93% positivity. Other reports by wet mount recorded 67.5% positivity ^[48] , and 75.8% positivity in 33 positive patients who were also positive by culture ^[49] . In contrast, lower rates of 36% ^[50] and 34.2% ^[51] were also recorded for wet mount as compared with culture, in addition to 33.6% ^[52] by wet mount of a total of 92 positive patients by culture. Therefore, Huppert et al. ^[53] concluded that wet mount alone is insufficient for the reliable diagnosis of trichomoniasis in women, although it is simple and cheap; in many health settings worldwide it is the preferred option for prompt diagnosis of trichomoniasis. This method saves time and enables patients to receive treatment on the same day and will reduce clinical overdiagnosis in women presenting with vaginal discharge ^[39] . However, it has its shortcomings. The sample should be examined promptly because T. vaginalis trophozoites are perishable in external conditions. During the process of deterioration, trichomonads lose motility, retract flagella, change morphology by becoming rounder, and thereafter become difficult to distinguish from similar-looking leukocytes ^[54],[55] .

Because of the wet mount limitations, culture remains more accurate for detecting the presence of T. vaginalis in patient samples. Routinely, 95% of patients were diagnosed by this method, and therefore it was considered the 'gold standard' ^[56] . The author advocated the use of culture when wet mount is negative and clinical suspicion is still present, and that it should also be performed to confirm a positive PAP smear in settings of low to intermediate prevalence. Accordingly, the primary purpose of staining is to optimize the visualization of key anatomic structures to facilitate accurate identification of the trophozoites. The use of staining methods is justifiable when it is not possible to use the wet mount method. In our study, Giemsa staining revealed 27.1% positivity compared with 35.3% positivity by culture; 83.3% were asymptomatic and 75% were symptomatic. The sensitivity of Giemsa stain method proved to be 76.7% compared with culture. In coordination with these results, Ojuromi et al. ^[47] reported 62 positive patients (73%) by stain compared with 85 positive by culture, whereas Radonjic et al. ^[15] reported 11 positive patients by stain out of 22 positive by culture method with 50% sensitivity, and Zaki et al. ^[49] reported 16 positive patients out of 33 (48.5%) by stain compared with culture. This controversy in results during the microscopic examination of Giemsa staining preparations may possibly be due to overlook or nonrecognition of trophozoites damaged during the process of staining. Overall, the Giemsa staining method is subjective and the result largely depends on the examiner's competence and qualification ^[57] .

AO staining is relatively simple to carry out, shows reasonable sensitivity and specificity but requires the use of a fluorescent microscope, which is not readily available in all settings ^[15] . In our study, 30.6% of patients were positive by AO stain compared with a total of 35.3% positive by culture method; of them, 27.3% were asymptomatic and 31.7% were symptomatic. Infection in the six asymptomatic patients diagnosed by culture was also positive by AO (100%). The sensitivity of AO proved to be 86.7% compared with culture. We were also able to show that AO staining method was more sensitive than wet mount as it diagnosed two patients who were missed by wet mount, which is in agreement with other reports ^[15],[20] that indicated a better sensitivity for AO staining than wet mount.

In the present study, 35.3% positive patients were detected by culture of the total 85 patients, conforming with the reports of 29.8% ^[47] and 30% ^[49] positivity by culture. The use of Diamond's culture was recommended as the most reliable and gold standard among conventional diagnostic methods in the detection of T. vaginalis ^[28],[58] . Moreover, subculture of the samples to increase its diagnostic efficacy was recommended. Presently, by culture we were able to detect six patients who were negative by wet mount, seven patients who were negative by Giemsa staining, and four patients who were negative by AO. These results are in compliance with the results of many authors who reported and confirmed that Diamond's culture remains reliable in the diagnosis of T. vaginalis infection [15, 28, 53, 59]. In general, culture methods are recommended by many authors who reported a sensitivity approaching 100%; in their view, as few as one parasite in the vaginal sample may flourish and be detected [28, 60, 61]. However, another report indicated that, to identify T. vaginalis by culture, it is necessary to have an inoculum of 300-500 trichomonads/ml ^[62] and that, if the sample contains an insufficient number of trophozoites, the result may appear to be false negative. Another opinion ^[63] stated that culture is not widely used as it is not available in most clinical laboratories and its specificity and sensitivity can be as low as 70-85% ^[50],[64] .

Because of the fact that reduced survival of trophozoites during transportation could negatively influence the sensitivity of culture, we inoculated specimens in prewarmed medium at once as recommended ^[15] . Despite the better sensitivity of culture technique, its expected result does not allow same day treatment, especially in asymptomatic patients, and the infection may continue to be transmitted. In many developing countries where the cost of return to the health facility can be substantial, patients may not bother to return for their culture results, leading to prolongation of infection and further transmission and complications. Partner notification efforts would also fail ^[53] .

On comparison, culture was found to be less sensitive than PCR [46, 48, 65, 66]. In the first report on the diagnosis of T. vaginalis in clinical samples by molecular method, TVA5 and TVA6 primers were used ^[22] . The technique was used to diagnose trichomoniasis in patients who had negative results by cultivation, as it was shown to detect T. vaginalis at a lower concentration than 300 trophozoites/ml necessary for culture ^[15] . In our study, on the whole, PCR was positive in 51.8% of all 85 patients. All positive patients by culture were also positive by PCR, which in addition recorded 14 extra patients who were negative by culture. Sensitivity of PCR was 100% as compared with culture, with 68.2% predictive value. In agreement with these results, it was reported that diagnosis of trichomoniasis by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs using molecular amplification methods ^[50] , having recorded a sensitivity of 98-100% compared with that of culture. Another report indicated that symptomatic patients with trichomoniasis were detected more by PCR (91.3%) than by culture (72.9%) or other routine methods ^[20] .

PCR also reportedly gave better performance than wet mount and culture [50, 60, 67, 68]. The one-tube nested PCR targeting the Tv-E650 repeat family of T. vaginalis was found simple, rapid, sensitive, specific, and accurate for the diagnosis of symptomatic and asymptomatic vaginal trichomoniasis when applied to vaginal discharge ^[51] . Moreover, it was reported that PCR alone is specific enough, so that there is no need for further confirmatory testing in the future, and that real-time PCR is the method of choice for the diagnosis of T. vaginalis infection as it showed 100 and 99.9% sensitivity and specificity, respectively ^[48] . In contrast, a lower sensitivity and specificity of 80.95 and 97.2%, respectively, for T. vaginalis detection by PCR was reported ^[15] . Another study indicated that PCR-based detection of T. vaginalis from vaginal swabs was not equivalent to culture where its sensitivity was 85.7-95.2%. In addition, the cost of PCR highly exceeded that of wet preparation and culture method ^[28] . It was stressed that, despite the fact that PCR detection of T. vaginalis proved to be rapid, specific, and very sensitive, culture of the organism still remains the gold standard for most laboratories [6, 59, 69].

Generally, PCR detection of T. vaginalis requires more technical skills and high costs. Molecular amplification techniques are currently in use in many laboratories for the detection of Chlamydia trachomatis and Niesseria gonorrhoeae infections; thus, PCR detection of T. vaginalis could easily be incorporated into the work flow of other diagnostic amplification procedures ^[50] . It must be emphasized that the results of PCR are available in a short time (1-2 days) and provide the highest sensitivity ^[50],[70] .

Conclusion

T. vaginalis infections are usually asymptomatic or can result in nonspecific clinical symptoms, which makes laboratory-based detection of this protozoan parasite essential for diagnosis and treatment. Before the molecular era, the concept was in using combined methods such as culture and AO staining that offer an advantage over the use of one test alone. Although the costs of PCR limit its wide application in the routine testing for trichomoniasis at the moment, PCR showed very high sensitivity and specificity when compared with the culture method, which is accepted as the 'gold standard'. The high sensitivity and specificity of PCR reported in this study offers a useful rapid screening tool and provides an excellent alternative for laboratory diagnosis of T. vaginalis. This could reduce spread and transmission of the infection, in particular from asymptomatic patients, minimize morbidity in women (cervical dysplasia, pelvic inflammatory disease, infertility, adverse pregnancy outcome), and decrease the healthcare costs.

Authors contribution

N.E. Nassef helped in performing the work and revising the manuscript. A.F. Afifi and A.F. Atia proposed the research idea and study design, preparation of the slides, stains, and culture. They helped in laboratory performance of the work, examined the slides, and wrote the manuscript. A.A. Basuni performed the PCR and helped in writing and revising the manuscript. M.F. Abo El-Nasr selected the patients, supervised the clinical evaluation of the patients, collected the samples, and revised the manuscript.

Acknowledgements

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