|Year : 2008 | Volume
| Issue : 1 | Page : 7-14
An update on Trichomonas vaginalis
Seema Sood, Arti Kapil
Department of Microbiology, AIIMS, New Delhi, India
Department of Microbiology, AIIMS, Ansari Nagar, New Delhi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Trichomonas vaginalis is a parasitic protozoan that is the cause of trichomoniasis, a sexually transmitted disease (STD) of worldwide importance. Recent data have shown that the annual incidence of trichomoniasis is more than 170 million cases worldwide. In fact, the World Health Organization (WHO) has estimated that this infection accounts for almost half of all curable sexually transmitted infections. The actual burden of the disease remains unknown in India. As per the published literature, the prevalence of T. vaginalis ranges from 0.4-27.4% in women and 0.0-5.6% in men. Although T. vaginalis is the most common cause of nonviral STD, the exact mechanism of its pathogenesis has not been clearly elucidated. Standard teaching is that trichomoniasis is an important cause of vaginitis in women. The prevalence and spectrum of disease in males are less well characterized; the infection appears to usually be asymptomatic, but it has been suggested as an increasingly important cause of nongonococcal urethritis. The laboratory plays a key role in the diagnosis of this infection. The time-honored approach has been microscopic evaluation by wet mount method. The broth culture method is the 'gold standard' for diagnosis of trichomoniasis and detects twice as many infections as the wet mount method. The drug of choice is metronidazole or tinidazole. For long it has been considered a 'minor' STD. Recent literature documents that women infected during pregnancy are predisposed to premature rupture of membranes, premature labor, and low-birth-weight infants. Further, it may amplify HIV transmission. Therefore, the identification of this common treatable sexually transmitted infections offers a precious and much needed additional strategy for AIDS prevention.
Keywords: Nongonococcal urethritis, sexually transmitted disease, Trichomonas vaginalis , wet mount
|How to cite this article:|
Sood S, Kapil A. An update on Trichomonas vaginalis. Indian J Sex Transm Dis 2008;29:7-14
| Introduction|| |
Trichomonas vaginalis is a parasitic protozoan that is the cause of trichomoniasis, a sexually transmitted disease (STD) of worldwide importance. Although the disease was first described as a clinical entity by Hohne in 1916,  the etiological agent, T. vaginalis , was first identified by Donne' who visualized motile microorganisms in the purulent, frothy leukorrhea of women presenting with vaginal discharge and genital irritation.  However, research on this organism did not begin until the 20 th century. In the 1960s and 1970s, the research focused on biochemical tests and microscopic examination to understand the growth characteristics and behavior of the organism. It was not until the 1980s that immunologic methods and molecular biology techniques became available and were applied to study the pathogenesis and immunology of this organism.
The life cycle of T. vaginalis is still poorly understood. Like many other protozoan parasites, it is known to exist only as a trophozoite and lacks a cystic stage. It is a primitive eukaryotic organism that in its carbohydrate and energy metabolism shows remarkable similarity to anaerobic bacteria.  It lacks mitochondria and instead uses the hydrogenosome to accomplish fermentative carbohydrate metabolism with hydrogen as the electron acceptor.
T. vaginalis is an obligate parasite in that it lacks the ability to synthesize many macromolecules de novo, particularly purines, pyrimidines, and many lipids. These nutrients are acquired from the vaginal secretions or through phagocytosis of host and bacterial cells.  Culture media for T. vaginalis therefore need to include all the essential macromolecules, vitamins, and minerals. In particular, serum is essential for the growth of trichomonads, since it provides lipids, fatty acids, amino acids, and trace metals.  In vitro , it grows optimally at a pH of 6.0-6.3,  although it can also grow through a wide range of pHs, especially in the changing environment of the vagina.
| Epidemiology|| |
Trichomonal infection has been encountered in every continent and climate and has no seasonal variability. It has a cosmopolitan distribution and has been identified in all racial groups and socioeconomic strata. Recent data have shown that the annual worldwide incidence of trichomoniasis is more than 170 million cases.  In fact, the World Health Organization (WHO) has estimated that this infection accounts for almost half of all curable sexually transmitted infections.  The incidence of trichomoniasis is as high as 56% among patients attending STD clinics. 
The actual burden of the disease in India remains unknown. Incidence figures are not reliable as facilities for diagnosis and treatment are inadequate. Only prevalence data are available from ad hoc surveys in population groups that are not necessarily representative of the total population. The majority of studies have thus far focused on examining prevalence rates in women, and even these data are relatively patchy and incomplete. Data on STD prevalence in men is lacking, especially for men in the 'general population.' As per the published literature, the prevalence of T. vaginalis ranges from 0.4-27.4% in women and 0.0-5.6% in men. 
Humans are the only natural host for T. vaginalis . The incubation period of the disease is typically 5-28 days. The trophozoite is transmitted from person to person through sexual intercourse. Nonsexual transmission of the disease is rare.  Asymptomatic infection rates are as high as 50% in women. Infected men are usually asymptomatic and the infection is usually self-limiting; diagnosis is thus often difficult.
| Clinical Manifestations|| |
Standard teaching is that trichomoniasis is an important cause of vaginitis in women, but that male sexual partners experience little or no morbidity. However, there is slowly growing literature suggesting the contrary in men  Signs of infections in symptomatic women include vaginal discharge (42%), odor (50%), edema or erythema (22-37%), and colpitis macularis, i.e., strawberry cervix (a clinical sign). Other complaints may include dysuria and lower abdominal pain. Nearly half of all women with T. vaginalis are asymptomatic. 
The prevalence and spectrum of disease in males are less well characterized; the infection appears to usually be asymptomatic, but it has been suggested to be an increasingly important cause of nongonococcal urethritis (NGU). 
Nongonococcal urethritis: Urethritis in men has been categorized historically as gonococcal or nongonococcal. Chlamydia trachomatis was long considered to be the cause of the largest subgroup of diagnosable cases of NGU, followed by Ureaplasma urealyticum . However, the rapid changes in STD epidemiology and the development of new diagnostic tests, in particular, the development of the noninvasive urine-based nucleic acid amplification tests, warrant a comprehensive review of this literature. 
On extensive review of the literature, surprisingly, chlamydia was found to account for a relatively small proportion of cases of NGU (15-31%). ,,, Ureaplasma urealyticum was detected in 9-22% of cases. Therefore, even under the best circumstances, up to two-thirds of Gram's stain-documented NGU is not due to these organisms. These findings have led to the consideration of other possible etiologies for NGU, which include Mycoplasma genitalium and T. vaginalis infection. However, the lack of a sensitive diagnostic test for T. vaginalis in most clinical settings has prevented more frequent identification of trichomoniasis as one of the etiologies of urethritis.  Recent studies have shown that T. vaginalis was isolated from 12-18% cases of acute nonchlamydial NGU and in 14.7% of patients with persistent urethritis. , In India, 2.5% of NGU patients were found positive for T. vaginalis . 
Taken together, this slowly growing body of data suggests that T. vaginalis represents an important consideration in the differential diagnosis of urethritis. T. vaginalis is an important cause of urethritis in sexually active men, especially in older men with urethral symptoms or inflammation but with little or no evidence of discharge on physical examination. Trichomoniasis is 'no longer a minor STD'  and consideration should be given to including specific treatment of trichomonas as initial treatment for NGU. 
The epidemiology of NGU is changing, with a decline in the proportion of cases resulting from chlamydial infection. Differentiation between chlamydial and nonchlamydial urethritis is important because of different public health approaches to control, such as partner notification and management of treatment failure. Many organisms implicated in nonchlamydial NGU, such as Mycoplasma genitalium and Ureaplasma urealyticum , are also found as commensals. Thus, sexual transmission cannot always be assumed. Most experts believe that partner referral is not routinely indicated in nongonococcal, nonchlamydial urethritis. The question then arises as to the need for new terminology for defining urethritis, i.e., gonococcal, chlamydial, and nongonococcal/nonchlamydial urethritis. 
According to the current recommendations (2006) of the Centers for Disease Control and Prevention, persons who have persistent or recurrent urethritis can be re-treated with the initial regimen if they did not comply with the treatment regimen or if they were reexposed to an untreated sex partner. In addition, a T. vaginalis culture should be performed using an intraurethral swab or a first-void urine specimen. If the patient was compliant with the initial regimen and reexposure can be excluded, the following regimen is recommended: metronidazole or tinidazole plus azithromycin (if not used for the initial episode). 
| Complications|| |
Although the incidence of trichomoniasis far exceeds that of gonorrhea and chlamydia, it is not a public health priority. Prevention of trichomoniasis has not been a priority due to lack of understanding of its public health implications and lack of resources.  For long it has been considered a 'minor' STD. It has been seen that women infected during pregnancy are predisposed to premature rupture of membranes, premature labor, and low-birth-weight infants. , Further, it may amplify HIV transmission. ,, The natural history of this organism, including its often symptomless nature and protracted carriage, play an important role in HIV transmission dynamics.
The organism typically elicits an aggressive local cellular immune response, with heavy infiltration of leucocytes, even in symptom-free patients. In addition, in about 50% of infected women, punctate hemorrhages can be observed. In an HIV-negative person, there are target cells available, as also access to blood stream. In an HIV-positive person, all this may expand the portal of exit for the virus and increase shedding of HIV-1 in the genital area. Thus, trichomoniasis may amplify HIV-1 transmission by increasing susceptibility in an HIV-1-negative person and the infectiousness of an HIV-1-positive patient. Therefore, the identification of this common treatable sexually transmitted infections (STI) offers a precious and much needed additional strategy for AIDS prevention.
| Pathogenesis|| |
Although T. vaginalis is the most common cause of nonviral STD, the exact mechanism of its pathogenesis has not been clearly elucidated. The host-parasite relationship is very complex, and the broad range of clinical symptoms cannot likely be attributed to a single pathogenic mechanism. Many mechanisms are thought to be involved and all the pathogenic mechanisms (i.e., contact-dependent, contact-independent, and immune response) are probably important in the virulence of this disease. 
The adhesion of the parasite to the epithelial cells seems to be mediated by four adhesion proteins: AP65, AP51, AP33, and AP23,  which act in a specific receptor-ligand fashion, dependent on time, temperature, and pH. Gene expression of the four adhesion proteins is coordinately upregulated at the transcriptional level by iron.  Little is known about the host cell receptors to which the parasite adhesion molecules bind, although there is some evidence that laminin may be a target for trichomonad adhesion.  Adherence, however, does not correlate directly with virulence, since virulent strains isolated from symptomatic patients exhibited wide differences in their ability to adhere to host cells.  On the other hand, the hemolytic activity of the parasite appears to be correlated with virulence.  Since the ability to synthesize lipids is lacking in T. vaginalis , erythrocytes may be a prime source of the fatty acids that are needed by the parasite. In addition, iron, which is an important nutrient for the parasite, may also be acquired by lysis of RBCs. Also, a variety of hydrolases have been described in T. vaginalis , with cysteine proteinases being particularly prevalent. 
The contact-dependent mechanisms described above play an important role in the pathogenesis of the disease. Besides these, there are contact-independent mechanisms. There are reports of other parasite products, described as cell-detaching factors (CDF), that are released by the parasite and are known to have trypsin-like activity. CDF, a 200-kDa glycoprotein, is heat and acid labile and its activity is pH dependent.  This is of clinical relevance since the normal pH of the vagina is 3.8-4.2 but is greater than 5.0 in the presence of trichomoniasis. The rise of vaginal pH during trichomoniasis may therefore be crucial in the pathogenesis of the disease. Further, the production of CDF has been shown to decrease in the presence of estrogen. This finding may explain some of the etiology of the disease, i.e., the worsening of symptoms around the time of menses, when estrogen levels are at their lowest.
The rise in vaginal pH is accompanied by a concomitant reduction (or complete loss) of lactobacilli. The relationship between protective lactobacilli and T. vaginalis is not completely understood. In addition, T. vaginalis has numerous ways of evading the immune system, which include evasion of complement-mediated destruction,  molecular mimicry,  and the ability to coat itself with host plasma proteins. 
The roles played by pH and hormones in trichomoniasis may explain the observation that the symptoms of the disease are often worse during menstruation.  The presence of menstrual blood creates a rich milieu for T. vaginalis reproduction, creating a higher pH than is normally found in the vagina. Additionally, the blood makes available increased amounts of iron, which enhances the ability of the T. vaginalis to attach to the vaginal epithelium. 
The presence of double-stranded RNA (dsRNA) viruses ( T. vaginalis RNA virus; TVV) in trichomoniasis was found to correlate with variation of the expression of certain surface antigens, and loss of the dsRNA accompanied loss of antigen expression.  However, the precise role of dsRNA viruses in the pathogenesis of trichomoniasis remains to be determined.
Experimental studies have shown that female animals are more susceptible to infection than their male counterparts.  T. vaginalis infection in males is generally mild or asymptomatic. The oxidative nature of the male genital tract is hypothesized to be inhibitory to certain pathogenic factors of the protozoan.  Zinc, in prostatic fluid, is also cytotoxic to the parasite.  In contrast, the vagina is a reducing environment, which may contribute to the activation of some pathogenic mechanisms of T. vaginalis .  Further, for understanding the strain differences in the virulence of the parasite, there are no well-documented physical or biochemical determinants of virulence factors on the basis of which strains can be classified.  No clear-cut zymodeme pattern has been assigned to strains of T. vaginalis  and restriction fragment length polymorphism analysis  could not clearly differentiate isolates from symptomatic and asymptomatic women.
| Diagnosis|| |
The classic symptoms associated with infection by T. vaginalis include a yellowish-green frothy discharge, pruritis, dysuria, dyspareunia, and the 'strawberry cervix,' which is characterized by punctate hemorrhagic lesions.  However, diagnosis cannot be readily made solely on the basis of clinical presentation for several reasons: (i) the clinical symptoms may be identical to those seen with other STDs, , (ii) the classic 'strawberry cervix' is seen in approximately only 2% of patients, and (iii) the characteristic frothy discharge is seen in only 10% of women with T. vaginalis .  If these classic features alone are used for the diagnosis of trichomoniasis, 88% of infected women will not be diagnosed and 29% of uninfected women will be mistakenly diagnosed as having T. vaginalis infection.  Therefore, the laboratory plays a key role in the diagnosis of this infection. Accurate diagnosis is essential, since it will lead to appropriate treatment and will facilitate the control of the spread of T. vaginalis infection.
The time-honored approach for the diagnosis of trichomonal infection has been microscopic evaluation by the wet mount method, a procedure first described by Donne' in 1836.  The presence of trichomonads is determined by the characteristic size (10 × 7 µm) of the organism, its shape, and the characteristic quivering / twitching motility. This procedure, however, detects only 35-80% of the cases, depending on the expertise of the microscopist. , Our study has demonstrated that the sensitivity of wet mount is 55%.  It is already established that a minimal concentration of 10 4 organisms per milliliter of vaginal fluid appears to be necessary for identification of the protozoan by wet mount.  Since the protozoa lose their distinctive motility on cooling to room temperature,  a microscope and an experienced microscopist must be readily available in the clinical setting.
In men, wet mount preparation is insensitive, and culture testing of urethral swab and urine is required for optimal sensitivity. The broth culture method is the 'gold standard' for the diagnosis of trichomoniasis; it is simple to interpret and requires as few as 300-500 trichomonads / ml of inoculum to initiate growth in culture.  Culture detects twice as many cases of trichomonas infections as the traditional wet mount method; our study also showed that culture detected 45% more positives than the wet mount.  However, there are inherent limitations to diagnosis by culture.  A period of 2-7 days is necessary before T. vaginalis can be identified in cultures, during which time infected patients may continue to transmit the infection. Also, no culture system is widely available to clinicians. Although several commercial liquid media are available for this purpose, Diamond's medium is considered the 'gold standard'. The InPouch TV culture system has been found to be as reliable as Diamond's medium in detecting T. vaginalis .  The InPouch TV is a double-pouched container made of soft, transparent plastic. The top pouch is inoculated with genital secretions suspected of having trichomonads and the specimen is pushed down into the bottom pouch, which serves as a container for the culture broth during the subsequent incubation. Although the combination of culture and wet mount examination remains the standard approach for detecting T. vaginalis in patient samples, InPouch does offer some distinct advantages: it is simple; urine can be used instead of urethral specimen; once the specimen is placed by the clinician into the InPouch chamber, microscopic observation can be made directly through the bag as the bag can be used as a slide on the stage of the microscope (this reduces chances of contamination); it can be conveniently transported from the site of collection to the laboratory; it can be stored at room temperature; and its cost is comparable to the ordinary culture tube.
The cell culture technique uses a variety of cell lines to isolate T. vaginalis from clinical specimens. This technique has been reported to be superior to broth culture and wet mount examination since it is able to detect the protozoa at a concentration as low as 3 organisms / ml. However, the cell culture technique is not routinely performed as it is expensive and not convenient for rapid diagnosis. 
Besides these, a number of staining techniques using acridine orange,  Leishman,  periodic acid-Schiff,  and Fontana  have been used to improve the sensitivity of direct microscopy. Papanicolaou (Pap) staining may be the most practical approach for the detection of asymptomatic infections as a large number of asymptomatic women undergo routine cytologic screening. Unfortunately, it has been found to be insensitive and is the least specific method for diagnosis of trichomoniasis. 
The other tests available include the antigen detection tests and the nucleic acid-based tests. The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, Massachusetts), an immunochromatographic capillary flow dipstick technology (sensitivity 83% and specificity 99%) is available.  PCR-based tests for T. vaginalis in women do not appear to offer a diagnostic advantage; PCR appears to have a far greater sensitivity in the diagnosis of this infection in males. 
| Treatment|| |
Metronidazole (a-hydroxyethyl-2-methyl-5-nitroimidazole) and tinidazole are effective for treatment of T. vaginalis infection. Current CDC guidelines recommend that metronidazole be administered orally as 2-g single dose or in a dose of 500 mg twice a day for 7 days and tinidazole be administered orally as 2-g single dose. 
Mechanism of action: Metronidazole is a 5-nitroimidazole, a heterocyclic compound with a nitro group on the fifth position of an imidazole ring. It is derived from the Streptomyces antibiotic azomycin.  It is a small molecule and enters T. vaginalis via passive diffusion. The compound itself is inactive, but anaerobic reduction results in the formation of a cytotoxic nitro radical anion. It is reduced in the hydrogenosomes of T. vaginalis by the enzyme pyruvate: ferredoxin oxidoreductase (PFOR), with metronidazole acting as an electron sink by capturing the electrons from the reduced ferredoxin, which would normally be donated to hydrogen ions to form hydrogen gas in the hydrogenase reaction. The reduction of the nitro group is also important as the drug enters the cell by passive diffusion; the reduction thus enables more drug to enter the cell by creating a favorable concentration gradient as reduction proceeds intracellularly. The nitro radical is hypothesized to bind transiently to DNA, disrupting or breaking the strands and leading to cell death.
The recommended metronidazole regimens have resulted in cure rates of approximately 90-95%, and the recommended tinidazole regimen has resulted in cure rates of approximately 86-100%. Tinidazole has a longer half-life, is eliminated at a significantly lower rate, and reaches higher tissue concentrations in the genitourinary tract than metronidazole; in addition, T. vaginalis isolates have lower MLCs to tinidazole than metronidazole.
| Resistance|| |
Clinical resistance is said to be present when there is failure to cure the infection after at least two consecutive courses of metronidazole. Accurate figures on metronidazole-resistant trichomoniasis are not available. It is estimated that approximately 2.5-5% of all cases of trichomoniasis display some level of resistance to metronidazole.  Resistance can affect both aerobic and anaerobic mechanisms of metabolism.
In trichomonads resistant by aerobic mechanisms, possibly the transcription of the ferredoxin gene is reduced, thereby decreasing the ability of the cell to activate the drug. It is theorized that reduced ferredoxin expression would result in lowered activation of metronidazole and less drug trafficking into the cell. Additionally, the oxygen-scavenging pathways are affected. In anaerobic resistance, the activities of PFOR and hydrogenase are decreased or nonexistent. Decreased hydrogenase activity and the concomitant reduction in hydrogen production is a factor in the impaired oxygen-scavenging mechanisms in the hydrogenosome. Since oxygen is a highly efficient electron receptor, increased levels of cellular (hydrogenosomal) oxygen result in impaired reduction and activation of metronidazole. If metronidazole is not reduced, the concentration of the drug is the same in the intra- and extracellular environments, and no additional drug enters the cell. Additionally, in the presence of oxygen, a reduced nitro free radical may be oxidized back into the original drug and then reduced to become a superoxide anion. This process is known as futile cycling and results in only limited cell damage due to the superoxide anions rather than cell death due to nitro free radicals. 
If treatment failure occurs with the 2-g single dose of metronidazole and reinfection can be excluded, the patient can be treated with metronidazole 500 mg orally twice daily for 7 days or with tinidazole 2 g as a single dose. For patients failing to respond to either of these regimens, clinicians should consider treatment with tinidazole or metronidazole at 2 g orally for 5 days. Because the ferredoxin levels and metronidazole resistance are inversely related, metronidazole resistance is considered relative and not absolute and it can, to some extent, be overcome by higher dosage.
If these therapies are not effective, further management should ideally include determination of the susceptibility of T. vaginalis to metronidazole and tinidazole. There is also a need for using alternative therapies to avoid the development of drug resistance to metronidazole.
| Vaccines|| |
Two distinct T. vaginalis vaccine candidates have progressed to the stage of human clinical trials. The first, in the 1960s, involved a trial of 100 women with refractory trichomoniasis, with the subjects receiving intravaginal inoculations with increasing numbers of heat-killed T. vaginalis cells.  The other in the 1970s, i.e., SolcoTrichovac or Gynatren, was prepared from heat-inactivated lactobacilli. Clinical trials have yielded inconclusive data.  However, the existence of a successful Trichomonas foetus vaccine (whole-cell)  is encouraging in the search for a vaccine against T. vaginalis .
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