Sexually transmitted infections (STIs) remain a major public health concern. More than 30 pathogens can be transmitted through sexual intercourse, and nearly one million people are infected with a curable sexually transmitted pathogen every day ^[1][2][1,2]. The World Health Organization estimated an incidence of more than 377 million cases of chlamydia, gonorrhea, trichomoniasis, and syphilis in women and men during 2020 [3]. Although the incident cases of trichomoniasis are nearly 156 million [4], these epidemiological data may be underestimated due to the high number of asymptomatic patients [5], the low sensitivity of the preferred diagnostic methods used in many regions [6], and the fact that Trichomonas vaginalis infection is not a notifiable disease [7]. For all these, trichomoniasis has been included in the list of neglected parasitic infections (NPI) by the Center for Disease Control and Prevention (CDC) [8].

Trichomoniasis is characterized by a wide range of signs and symptoms associated with the inflammatory response triggered by the settlement of the parasite [9]. In women, about 75% of patients develop clinical manifestations. The most common are pruritus, local edema, erythema, dysuria, and/or a typical green, frothy, and malodorous vaginal discharge, among others ^[5][10][11][5,10,11]. In men, nearly 80% are asymptomatic; however, nongonococcal urethritis, epididymitis, or prostatitis may occur ^[5][10][5,10]. Although trichomoniasis has been considered as a “nuisance” infection ^[12][13][12,13], the complications and risks associated with this STI have led to its inclusion in the WHO Global Health Strategy on STIs for the period 2022–2030 [2]. T. vaginalis increases, by 1.5 times, the risk of acquiring HIV [14] but also favors its transmission due to the imbalance in the vaginal microbiome, the proinflammatory immune response, and the elevated vaginal pH [15]. In this scenario, coinfections with different urogenital pathogens are common among women with trichomoniasis, i.e., Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, human papillomavirus, or herpes simplex virus types 1 and 2 ^[16][17][16,17]. Other serious sequelae include pelvic inflammatory disease (PID) [2], adverse pregnancy outcomes ^[9][18][9,18], and infertility [9]. Moreover, several studies have associated trichomoniasis with an increased risk of cervical carcinogenesis ^[19][20][21][19,20,21]; notwithstanding, the association between T. vaginalis and prostate cancer remains under discussion ^[22][23][24][22,23,24].

This STI has been treated with metronidazole since 1959 [25]. Four decades later, the use of tinidazole was accepted ^[26][27][28][26,27,28], and recently the Food and Drug Administration has approved the use of secnidazole in the United States [29]. Likewise, in recent years, new recommendations on dosage in the treatment of infected women have been proposed to achieve a better rate of complete cure [28]. However, in spite of all this, almost 10% of clinical cases are resistant [30] and cross-resistance between 5-nitroimidazole drugs has been reported [31]. The absence of pharmacological alternatives to cope with treatment failure, hypersensitivity to 5-nitroimidazoles, or side effects ^[32][33][32,33] increases the risk of transmission and the development of chronic infection. Additionally, as trichomoniasis is highly asymptomatic and there are no diagnostic procedures to identify resistant and asymptomatic cases, the diagnosis based on syndromic management or microscopic identification of the parasite [34] hinders the correct management of the infected population. Therefore, the implementation of techniques with high sensitivity and specificity for use in routine and universal screening would reduce the incidence of this STI and, therefore, the risk associated with the acquisition of other pathologies, contributing significantly to this serious health problem.

2. Classical Diagnostic Procedures: Microscopy

Diagnosis of trichomoniasis has traditionally consisted of taking a specimen from the patient and examining it immediately under the microscope ^[35][36][44,45] to identify the characteristic morphology and motility of the trophozoite. The most reliable specimens for the diagnosis of female trichomoniasis include endocervical and vaginal swabs and urine ^[37][38][39][46,47,48], while in male patients are urine, urethral swabs, and semen ^[39][40][48,49]. Cervicovaginal and urethral specimens are obtained with cotton swabs or polyester sponges ^[35][36][37][44,45,46]. Today, wet mount microscopy is the fastest and most widely used method for diagnosing trichomoniasis in resource-limited areas ^[41][50]. This method can have a specificity of 100%; nevertheless, it must be carried out quickly enough and at a temperature that does not impair the viability of the trophozoite. It is important to note that sensitivity values may decrease depending on the time elapsed from sample collection to microscopic examination ^[42][41]. Furthermore, this subjective method achieves a sensitivity that, depending on the experience of the technician, can range between 35–80% in comparison with the culture method ^[43][44][45][51,52,53]. In addition, a delay in specimen transport reduces the motility of trichomonads, which affects the procedure’s sensitivity ^[46][47][54,55]. This method is the most efficient diagnostic test, but its reliability and sensitivity are not optimal ^[44][45][52,53]. This low potential sensitivity contributes to the underdiagnosis of the disease. Ideally, the saline wet mount preparations should be examined from the swab collected by the clinician, as well as inoculation of the samples immediately after collection in an appropriate culture medium ^[46][48][35,54]. Also, if samples are not immediately observed, they can be maintained in a suitable transport medium to avoid dehydration and redox potential changes. Stuart’s transport culture media and its modifications are the most recommended ^[49][50][51][56,57,58]. The average survival time in these transport culture media is approximately 24 h ^[52][59]. Inadequate transport or storage conditions may reduce the parasites viability and influence the wet mount technique sensitivity ^[42][41]. In this context, permanent staining techniques were developed as a complement to the direct examination of wet mount preparations ^[36][45].

2.1. Wet Smears

Microscopic observation of vaginal exudate diluted in saline is the routine procedure for the diagnosis of female trichomoniasis; however, epithelial cells and polymorphonuclear leukocytes in the samples may interfere with the parasite’s flagellar motility. The sensitivity of this technique is highly variable due to multiple causes, such as the type of sample, the number of viable organisms, and the delay between the obtention and the microscopic diagnosis, among others ^{[43][44][45][53][54]}[51,52,53,60,61]. Regarding male samples, Feinberg and Whittington observed a greater sensitivity when direct microscopy was used with urethral material than culture methods ^[53][60]; however, there are discrepancies in other reports ^[55][62].

2.2. Staining Techniques

Different stains have been developed to increase the sensitivity of direct examination. Stained smears can be preserved without loss of diagnostic reliability due to adequate fixation and be observed later ^[36][45]. The most frequently used stains include Papanicolaou, Giemsa, and acridine orange ^{[56][57][58][59][60][61][62]}[63,64,65,66,67,68,69]. Furthermore, less-well-known stains have also been tested, including Leishman, periodic acid–Schiff, and Fontana–Masson stains ^[60][61][62][67,68,69]. The Giemsa stain is perhaps the most accessible in the laboratory and has been used for more than 100 years in trichomoniasis diagnosis ^[63][70]. In these preparations, the nucleus of trichomonads stains purplish red and the cytoplasm is light red, pink, or bright blue, depending on pH, with a darker staining nucleus that may be oval or spindle-shaped. Sometimes, axostyle and flagella can be observed ^[64][71]. Generally, microscopic examination of Giemsa-stained smears is more effective in detecting infections than wet smear microscopy ^[65][66][67][72,73,74] and may have a sensitivity near that of culture ^[60][65][68][67,72,75]. Other stains such as safranin, methylene blue, and malachite green, which do not stain trophozoites, can act as counterstains ^[69][70][71][76,77,78]. Fluorescein can also be used to observe wet mount slides under an ultraviolet light microscope ^[72][79]. Thus, acridine orange for fluorescence-based detection of T. vaginalis has also been suggested by other authors as it exhibits a greater sensitivity than Giemsa staining but requires UV fluorescent light microscopy ^[38][73][47,80]. However, these staining methods have not been convincingly demonstrated to improve the detection rate of trichomonads in secretions and are not recommended for routine clinical diagnosis. In Papanicolaou smears of cervicovaginal material, T. vaginalis exhibits an ovoid structure and an approximate size of 10–30 μm with a greenish grey cytoplasm which contains very small eosinophilic granules, and the eccentric nucleus stains blue. The sensitivity and specificity of this stain vary depending on the experience of the microscopist ^[42][70][74][41,77,81] as shown in Table 1. The traditional diagnostic methodology is easy to perform; however, these techniques have the disadvantage of not being very sensitive and require careful observation by expert microscopists.

2.3. Culture

Liquid or broth culture of a clinical specimen (cervicovaginal, urethral, or urinary sediment) for microscopic observation has been considered the gold standard technique for the diagnosis of trichomoniasis, due to its sensitivity, simplicity, and the relatively low inoculum requirement (300 trichomonads/mL) ^[75][82]. Several media have been described for the T. vaginalis culture: Kupferberg, Kupferberg STS, Hirsch, Trichosel, Modified Diamond, Lash serum, or the most recent, called InPouch^® TV ^[76][77][78][83,84,85]. However, the most common are Diamond (TYM), modified Diamond, or Roiron^{® [42]}[41]. Diamond’s medium requires refrigeration at 4 °C for storage but should be at room temperature before specimen inoculation. Samples should be inoculated immediately into the culture medium, at least 1 h after collection, and incubated at 37 °C in anaerobic conditions (5% CO₂). This should be followed by daily examination for 3–7 days until viable trichomonads are observed ^[42][75][41,82]. Longer incubation times are often required in male specimens to allow the growth of a detectable number of organisms [6]. Thus, this methodology is simple and inexpensive, but requires the direct microscopic examination during a long period ^[40][49] in which infected patients may continue to transmit the infection ^[79][86]. Moreover, there are inherent limitations to culture diagnosis, e.g., culture contamination with vaginal microbiota (bacteria or yeasts) can be very frequent ^[80][81][87,88]. Nevertheless, sensitivity rates can rise to nearly 95% depending on the sample and the medium used (Table 1). To enhance the acceptance of culture diagnosis, a good procedure is the so-called delayed inoculation, a method that combines both techniques: first, the fresh sample for direct examination and, if negative, its incubation in culture medium for 2–5 days ^[82][89]. Regarding this, InPouch^® TV is a self-contained system which permits both immediate examination and culture in a single device of vaginal, urethral, and urine samples. The sensitivity is comparable to that obtained with wet smears and culture specimens ^[83][84][90,91]. The transparent oxygen-resistant plastic can be examined directly under the microscope, allowing daily examination of the specimen without removing it from its culture medium. InPouch^® TV can be kept at room temperature, and even inoculated pouches can remain at room temperature for up to 48 h before incubation at 37 °C ^[42][41]. Levi and coworkers demonstrated that the InPouch^® TV system was as sensitive as modified Diamond’s medium for T. vaginalis detection ^[85][92]. Borchardt and collaborators demonstrated that this system is more sensitive than modified Diamond’s medium or Trichosel medium ^[86][93]. However, InPouch^® TV continues to be a procedure that requires observation of the sample for several days, not a rapid diagnostic technique ^[40][42][41,49]. Therefore, it was necessary to develop and implement more rapid, sensitive, and specific tests that allow early diagnosis in at-risk populations and even the identification of asymptomatic cases. These new methods based on immunological and molecular tools, some of them POC techniques, are currently being implemented in many diagnostic services ^[84][91].