Parasite

Trichomonas vaginalis

Introduction

Trichomonas vaginalis (T. vaginalis, trichomonas, or “trich”) is the parasite responsible for trichomoniasis, the most common curable sexually transmitted infection worldwide. Although the symptoms of trichomoniasis are often mild or nonexistent, infection predisposes carriers to acquisition of other STIs, such as HSV-2 and HIV. Women with trichomoniasis are more likely to experience complications in pregnancy, as well as infertility and pelvic inflammatory disease. Because of these associated risks, trichomonas vaginalis has the potential to affect multiple aspects of reproductive health, and is emerging as a parasite of great public health importance.

History of Discovery

Trichomoniasis was first identified by Parisian physician Alexandre Donne in 1836, who described its “undulating motion” and “whiplike tail” (Hadju). Even at that early date, he also noted its concurrence with other diseases, such as gonorrhea and syphilis. He called the organism “trichomonas” because of morphological similarities to two other protozoa known at that time, tricodes and monas (Hajdu).

Agent (classification and taxonomy)

D. Scott Smith, Stanford University

Trichomonas vaginalis is an early protozoan—a unicellular eukaryote—in the same evolutionary group as parasites like toxoplasma gondii, giardia lamblia, and leishmania. Because t. vaginalis lacks mitochondria, it must have diverged from the majority of (mitochondria-containing) eukaryotes early on that phylogenetic branch—somewhere before kinetoplastids, which are the earliest protozoa known to use mitochondria to generate power in the form of ATP (Schwebke 2004)

Instead of mitochondria, trichomonas vaginalis produces ATP via a membrane-bound organelle called the hydrogenosome.

(A) trichomonad hydrogenosomes magnified 7500 times. (B) Sectioned hydrogenosomes showing a double membrane, magnified 75,000 times. (Bradley et al 1997)

The evolutionary origins of that organelle (and thus of trichomonas, itself), have been heavily debated.

Because hydrogenosomes contain enzymes normally found in anaerobic bacteria (prokaryotes), some scientists hypothesized that hydrogenosomes, and thus the trichomonas bug, were more evolutionary related to bacteria than to eukaryotes. However, hydrogenosomes and mitochondria also share many common characteristics, including a double membrane and a common means of protein import, suggesting that trichomonas is more closely related to mitochondria-using eukaryotes.

Hrdy et al (2004) and others provided increasing evidence in favor of the latter hypothesis by showing that the first few subunits of the respiratory chain in mitochondria also exist in trichomonads’ hydrogenosomes.

That buildup of evidence received final confirmation in January 2007 when the complete T. vaginalis genome was published, via the method of shotgun sequencing, by the Institute for Genomic Research.

Sequencing of the parasite’s complete genome also revealed that it is one of the largest we know of—about the size of the human genome. Much of that size is accounted for by repeated segments, which most likely reflect an expansion of genetic material due to a relatively recent evolutionary transition into the urogenital environment (Carlton et al 2007).

Morphology

T. vaginalis parasites can be 7-30 micrometers long, and normally appear pear-shaped when cultured. When attached to vaginal tissues, however, they may appear more amoeboid (CDC).

Trichomonads have five flagella—long “arms” or “tentacles” used for propulsion—four in the front, and one in the back. They have a barbed tail called an axostyle which helps them attach to vaginal tissue, and most likely causes the vaginal irritation associated with infection (trichomoniasis.org). The parasites’ nucleus is located at its wider front end (CDC).

(trichomoniasis.org)

Trophozoites obtained from an in-vitro culture, Giemsa stain (CDC)

Clinical Presentation in Humans

Although about one-third of women, and most men, are asymptomatic carriers of trichomonas vaginalis, symptoms may appear after an incubation period of 5-18 days (CDC).

In women, symptoms usually include inflammation of vaginal mucosa and vulvar irritation (vaginitis), along with pus-like discharge, vulvar and cervical lesions, and abdominal pain. Painful urination and painful sexual intercourse are also common (CDC). If symptoms appear in men, they are usually generalized and consist of urethritis, epididymitis, or prostatitis. (CDC).

In women, trichomoniasis can also be associated with more serious complications.

Multiple studies have linked trichomoniasis to adverse pregnancy outcomes. In a study of 13,816 women treated at university-affiliated hospitals in five US cities, Cotch et al (1997) found that pregnant women infected with t. vaginalis were significantly more likely to have a low birth weight infant and/or to deliver preterm. Treatment for trichomoniasis does not appear to have an effect on those outcomes: Klebanoff et al (2001), for example, found that metronidazole (one commonly used drug) does not prevent preterm delivery due to trich.

Trich has also been linked to infertility, pelvic inflammatory disease (PID), and cervical neoplasia (trichomoniasis.org). In a cross-sectional study, Cherpes et al (2006) found that women with trich were more likely to have PID; co-infection with HSV-2 (Herpes Simplex Virus) increased that likelihood. Trich has also been independently associated with HSV-2 acquisition; one study found that women with trich were almost four times as likely to acquire HSV-2 (Gottlieb et al 2004).

Trichomoniasis is a biological as well as a behavioral risk factor for HSV-2 as well as other STIs: Multiple studies have established an association between trich and HIV acquisition. A prospective study among women in Mombasa, Kenya found that trich increased risk of HIV-1 acquisition by over 50%. (McClelland et al 2007). The association holds in the United States, as well: Sorvillo and Kerndt (2005), for example, found high rates of trich infection among women with HIV-1 at a public clinic in Los Angeles.

There are a few different hypotheses about the biological relationship between trich and HIV. Because trichomonas elicits a local immune response, an HIV positive person’s infected CD4 cells concentrate at the site of infection in the genital area, and thus are more likely to be transmitted to a partner. Researchers also think that trichomonas may break down physical barriers against HIV by creating small hemorrhages in the vaginal tract through with the virus can enter (McClelland et al 2007).

Some research also suggests a relationship between trich and cervical cancer (Zhang et al 1995, Yap et al 1995). In a sample of 121 cervical cancer patients and 242 controls, Yap et al found trichomonas antibodies in the sera of 41.3% of patients. versus 5% of the controls.

Life Cycle

Trichomonas trophozoites have a simple life cycle. Only a trophozoite form exists (no cyst form has been identified), and it cannot survive for any significant period of time outside the human body, so the parasite has no reservoirs or vectors.

Trichomonads live either in the female lower genital tract or in the male urethra and prostate, where they replicate asexually, by binary fission. They are then transmitted from an infected person to a partner via sexual intercourse (CDC, Schwebke and Burgess 2004). After an incubation period of 5-18 days, an infected person may begin to show symptoms, although they can also remain asymptomatic.

(CDC)

Transmission

Trichomonas is considered a sexually transmitted infection (STI) because it is primarily transmitted through sexual intercourse—either oral, anal, or vaginal. Sex between women, or between men and women, can effectively transmit the parasite; it is unusual for it to be transmitted man-to-man (trichomoniasis.org).

An infected mother can also pass trichomonas to her child during birth, where it can infect a newborn’s genital area or lungs. This occurs in 2-17% of cases, according to several different studies (Schwebke and Burgess 2004).

Very rarely, trichomonas vaginalis can be transmitted through contaminated specula or toilet seats. However, trichomonas dies quickly in a dry environment, so this mode of transmission is unlikely (Whittington 1957).

Diagnostic Tests

The symptoms associated with trichomoniasis are not sufficient for diagnosis, not only because it is often asymptomatic, but because symptoms, when they do occur, can be very generalized. However, a variety of additional diagnostic methods can be employed to diagnose trichomoniasis:

Wet mount

The quickest and easiest method of trich diagnosis is via a wet mount: a vaginal, urethral, or (for men) prostatic fluid sample is examined under a microscope for the presence of parasites. They will be identified by a jerky, whole-body movement or by the beating of their flagella while at rest. Wet mounts need to be performed within 20 minutes of collection, before the parasites lose their motility. trichomonads under a wet mount are about the size of white blood cells, clear, and teardrop-shaped (see image below).

Additional indicators that can help confirm the presence of trichomonads in a wet mount slide are (1) an increased level of white blood cells; (2) an elevated vaginal pH (greater than 4.5); and (3) a positive “whiff test” (addition of potassium hydroxide to vaginal fluid produces an amine odor). (CDC, Schwebke 2004)

Trichomonas vaginalis on a wet mount slide; trichomonas and a white blood cell identified (USC Keck School of Medicine)

Culture

Identifying trichomonas vaginalis via culture is considered the “gold standard,” or most sensitive method, for detection. It takes much longer than a wet mount diagnosis: results are not available for 3 to 7 days.

Diamond’s medium is standard for culture, but studies have shown a manufactured culture kit (the InPouch TV kit) to be an equally accurate, easier to use alternative (Draper 1993). Some tests have even found the InPouch technique to be more sensitive (Borchardt et al 1997).The pouch has two chambers, allowing for wet mount observation followed by incubation for three days. If no trichomonads are observed after 3 days, the test is presumed negative.

Trich diagnosis has been explored with a variety of other methods, as well.

Several different PCR-based tests have been developed and tested, with reported sensitivities of 85-100%. So far, it does not appear to have any significant diagnostic advantage over wet mount and culture (Schwebke 2004), although refinement of the tests appaear to be slowly improving this method. A recent NIH study comparing InPouch TV and PCR reported a sensitivity of 97% for the PCR test, versus a culture sensitivity of 70%, and a wet mount sensitivity of 36% (Madico et al 1998).

Nucleic Acid Amplification Tests (NAATs) are another new method currently being pioneered. Nye et al 2009 compared NAAT to traditional diagnostic methods, and found that the test had improved diagnostic accuracy compared to wet mount or culture, as well as being faster than culture. Prevalence in women ranged from 16.2% from wet mount diagnosis, to 28.7% by NAAT.

New “point of care” diagnostics—rapid test kits—are also being developed for trichomoniasis. Two new tests, the OSOM Trichomonas Rapid Test and the BVBlue Rapid Test, both designed by Genzyme Diagnostics in Cambridge, Mass, have been approved by the FDA and are currently undergoing further clinical trials at the university of Pittsburgh. As of now, testing shows that they are just as accurate as existing standard tests, but take approximately 10 minutes, as opposed to days, to get results (Huppert et al 2005).

Other research, rather than developing new tests, focuses on improving the abilities of currently used diagnostic tests. Shafir et al (2007) increased the viability of t. vaginalis in urine by processing samples through a filter before placing them in culture medium, increasing the sensitivity of that existing diagnostic. Filtering removed trichomonad-damaging impurities out of the urine, including other medications, STIs, or organisms.

Trichomonas can also be diagnosed via an existing pap smear test with a sensitivity of 60% and a specificity of approximately 95% (Schwebke 2004).

Management and Therapy

Two drugs are commonly used to treat trichomoniasis. One is Metronidazole (brand name Flagyl), and the other is Tinidazole. Both are similarly-structured nitroimidazoles, a class of heterocycle that is often used to treat bacterial and parasitic infections. Both have a high cure rate. Because trichomoniasis is a sexually transmitted infection (STI), an infected person’s sexual partners must also be treated in order to eliminate the infection (CDC).

The drugs operate similarly within the body. They are small molecules, and so are able to pass through the t. vaginalis membrane. They enter the parasite in an inactive form, but once inside their nitro groups (indicated by the arrow on the diagram below) are reduced (they gain electrons via chemical reaction), leading to the generation of nitro radicals which then bind to and damage the parasite’s DNA and interior structures, leading to death of the parasite (Sood and Kapil 2008, Megraud et al 2001).

The reduction of the nitro group serves another purpose, as well: because reduction makes the molecule inside the trichomonad different from the one outside, a concentration gradient is established across the trichomonad’s membrane that lets more of the drug enter the cell, where it can do more damage to the parasite (Sood and Kapil 2008).

Arrow points to the nitro group on metronidazole. (Megraud et al 2001).

Recommended dosages

Drug	Adult dosage	Pediatric dosage
Metronidazole	2 g PO once OR 500 mg bid x 7d	15 mg/kg/d in 3 doses x 7 d
Tinidazole	2 g PO once	50 mg/kg once (max 2 g)

(The Medical Letter)

A single 2 gram dose is usually preferred over multiple doses, both because then there is no need to worry about patient compliance, and because less total drug is required (meaning less potential for side effects). Again, to prevent reinfection, sexual partners should be treated simultaneously with the same dosage as the infected person.

Metronidazole can be administered intravenously as well as orally, although (for logistical reasons) this is not common. If a patient suffers from side effects when taking oral metronidzole, administering the drug intravenously may reduce them (Cudmore 2004).

More about metronidazole

(Wikipedia)

Metronidazole was developed in 1959, and quickly approved for use by the United States in the early 1960s (Cudmore 2004). It is very effective at treating trichomoniasis, with cure rates reported at 90-95% (Sood and Kapil 2008).

Side effects

Normally, metronidazole has few or no side effects. When they are reported, they are usually mild, and include nausea, vomiting, headache, insomnia, dizziness, drowsiness, rash, dry mouth, and, if taken orally, a metallic taste. More serious side effects are rare, and include eosinophilia, leucopenia, palpitation, confusion, and peripheral neuropathy (Cudmore 2004).

Multiple studies (Klebanoff et al 2001, Kigozi et al 2003) have found that the use of metronidazole during pregnancy may have particular negative effects. Kigozi et al, for example, found that children of women treated for t. vaginalis with metronidazole had an increased risk of low birth weight, an increased preterm birth rate, and a higher 2-year mortality rate than children of trichomonas-infected women who were NOT treated with the drug. Researchers hypothesize that metronidazole can have these negative effects during pregnancy because it can cross the placental barrier. For these reasons, although a single 2g dose of metronidazole is approved for pregnant women by the CDC, metronidazole is also classified by the FDA as a “class B risk factor” posing “possible but unconfirmed” risk to a fetus. The FDA also recommends a 24 hour interruption in breast feeding after taking metronidazole, because a small amount of the drug is secreted in breast milk. This recommendation is controversial, however, because interrupting breast feeding for such a long period of time is also known to have deleterious consequences (Cudmore 2004).

Drug resistance

“Resistance” to trichomoniasis treatment is defined as failure to cure the infection after at least two consecutive courses of metronidazole (Sood and Kapil 2008). T. vaginalis parasites in at least 5% of clinical cases exhibit resistance.

Resistance can be achieved through a number of potential mutations. Most likely, resistant trichomonads reduce transcription of a gene or genes coding for enzymes that once helped activate the drug when it was inside the parasite, such as ferridoxin, malic enzyme, or hydrogenase (Schwebke and Burgess 2004).

That resistance can often be overcome by more medication (Sood and Kapil 2008), so if treatment fails, simply increasing dosage—to 500 mg metronidazole orally twice daily for 7 days, or to 2g oral tinidazole—is recommended. If that fails, a further dosage increase is recommended, to tinidazole or metronidazole 2g orally for 5 days. (Sood and Kapil 2008). The tradeoff, of course, is an increased risk of side effects from a higher and more toxic dose (Cudmore 2004).

Tinidazole

Tinidazole was recently licensed for use in the United States, and appears to have a successful but slightly more variable cure rate than Metronidazole (86-100%) (Sood and Kapil 2008).

It differs from metronidazole in that it is eliminated from the body more slowly—it has a longer half-life—and in that the concentrations of the drug found in vaginal secretions are closer to levels found in serum, meaning that it is delivered more effectively to the target area of the body. For this reason, tinidazole works at lower doses than metronidazole, leading to milder side effects (Cudmore 2004, Sood and Kapil 2008). Nontheless, it should be taken with food to minimize GI problems (The Medical Letter).

Potential for a vaccine

The body’s natural immune response to t. vaginalis infection does not provide long-lasting protection from the parasites. After the immune system fights a trichomonas invasion, antibody levels quickly decline, and by 6 to 12 months, the body is left with no acquired defenses against another trichomonas infection (Cudmore 2004). Thus, a trichomonas vaccine would be incredibly useful in providing a more lasting immune response to the parasite.

Research on the body’s immune response to trichomonas has flagged some potential vaccine development strategies. For example, one study identified a particular protein in t. vaginalis, the 115-kDa-actin protein, against which women develop an immune response (Addis et al 1999). It may be possible to elevate this particular response via a vaccine, thus boosting the body’s natural reaction to trichomonas invasion.

Additionally, because different strains of t. vaginalis share this protein, it may be possible to develop a vaccine that protects against a number of different t. vaginalis strains at once (Garber et al 1986).

To date, two vaccines have been developed and progressed to human clinical trials, although neither have made it to the general market.

The first, in the 1960’s, administered intravaginal inoculation with heat-killed t. vaginalis cells to 100 women in attempt to induce immunity. The second, in the late 1970’s, was developed from heat-killed abnormal lactobacilli isolated from women with trichomonas infection. Although both exhibited some success, neither have been further pursued (Cudmore 2004).

More recently, Abraham et al (1996) studied the effect of trichomonas immunization in a mouse model. Mice were immunized subcutaneously with heat-killed t. vaginalis; this appeared to confer long-lasting immunity over control saline injections or simple treatment with metronidazole (which conferred only a temporary immune response).

Epidemiology

Trichomonas vaginalis is found worldwide (CDC) and is the most common curable sexuallty transmitted infection, with an estimated 174 million new cases a year, the majority (154 million) of which occur in resource-limited settings (trichomoniasis.org). Its epidemiology and spread are greatly affected by the fact that it is often asymptomatic, and can be passed through intercourse without either partner’s knowledge.

Only inconsistent data on t. vaginalis prevalence exists. Most data comes from pregnant women, who are the most likely carriers of trich to have access to health care and to receive testing.

The prevalence of trichomoniasis in resource-limited settings worldwide is estimated to be between 3.2-34% in women, and 6.3-11% in men. (Johnston and Mabey 2008).

In Latin America, WHO prevalence estimates among pregnant women ranged from 2.1% in Brazil to 27.5% in Chile (WHO). In Africa, they ranged from 9.9% in the Central African Republic to 41.4% in South Africa (WHO).

In the United States, prevalence was recently estimated to be 3.1% among women of reproductive age, although this varied greatly between racial groups, with the main risk group being African-American women, who had a prevalence that was 10.3 times higher than that among white and Mexican-american women (Sutton et al 2007). There are multiple reasons why this may be the case, including a high prevalence of trich infection among partners, a decreased use of barrier protection during sex (supported by research on racial/ethnic differences in condom use), an increase in practices such as douching, lack of access to health care, or greater noncompliance with treatment (Sorvillo et al 2001).

Other high-risk groups include prison inmates, drug users, and sex workers—those at higher risk of contracting STIs in general (Johnston and Mabey 2008).

Public Health and Prevention Strategies

For a long time, trichomoniasis was considered an insignificant infection—it was asymptomatic, and when symptoms did show, they were mild. However, as research uncovers an increasing number of ways in which trich is linked, biologically (as well as in terms of sexual behavior), to pregnancy complications as well as to other STIs, researchers have started to recognize its importance to reproductive health as a whole. Because trich is so easily treated compared to many of the complications it is associated with, it can play a key role in public health interventions (Johnston and Mabey 2008), particularly among high-risk groups.

Interventions designed specifically to treat and resolve cases of trichomoniasis can also prevent other, more serious complications.

As mentioned earlier, numerous studies have shown that trichomonas amplifies HIV transmission rates by increasing CD4 cell density and expanding areas for HIV to mechanically enter the body. Studies suggest that the amplification may be twofold (Sorvillo et al 2001) or 1.5-fold (McClelland 2001). This makes trichomonas treatment a low-cost, potentially key public health measure for critically-needed HIV prevention.

Treatment of trichomoniasis may help reduce incidence of PID, pregnancy complications, and HSV-2. Routine testing at clinics, or (where prevalence is high) more proactive community-based screening and treatment, as well as efforts to treat asymptomatic partners, are all measures that can be taken to reduce incidence of trich and its complications.

Aside from treatment for trichomoniasis, however, it is important to note that the same basic public health interventions that protect against all STIs will protect against trichomonas, HIV, HSV-2, and others. Promotion of monogamy, testing, and condom use are standard public health measures worldwide, but female-controlled alternatives to condoms should be considered where appropriate, such as female condoms or new technologies like microbicides, some of which are undergoing clinical trials. A recent study evaluated the effectiveness of Sapindus saponins, a component of an Indian herbal contraceptive marketed in India, and found that it decreased trichomonad concentrations in infected patients (Tiwari et al 2008). Innovative new treatments such as these could prevent new cases of trichomonas, circumvent issues of resistance to current drugs, and serve to decrease the prevalence of trichomonas and related STIs worldwide.

Web Links

· http://www.dpd.cdc.gov/dpdx/hTML/Trichomoniasis.htm

o CDC’s “Parasites and Health” site, with information on the trichomonas parasite, its geographic distribution, clinical features, laboratory diagnosis, and treatment

· trichomoniasis.org

o Information (for the general public) on the parasite and disease

· http://www.youtube.com/watch?v=9B1PFJXuJUs&feature=PlayList&p=40E9D73AF0C42874&index=0&playnext=1

o YouTube video of a trichomonas wet mount—trichomonads exhibit characteristic motility.

References

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