Trichomoniasis: evaluation to execution

Abstract

Trichomoniasis is the most common sexually transmitted disease, caused by a motile flagellate non-invasive parasitic protozoan, Trichomonas vaginalis (T. vaginalis). More than 160 million people worldwide are annually infected by this protozoan. T. vaginalis occupies an extracellular niche in the complex human genito-urinary environment (vagina, cervix, penis, prostate gland, and urethra) to survive, multiply and evade host defenses. T. vaginalis (strain G3) has a ~160 megabase genome with 60,000 genes, the largest number of genes ever identified in protozoans. The T. vaginalis genome is a highly conserved gene family that encodes a massive proteome with one of the largest coding (expressing ~4000 genes) capacities in the trophozoite stage, and helps T. vaginalis to adapt and survive in diverse environment. Based on recent developments in the field, we review T. vaginalis structure, patho-mechanisms, parasitic virulence, and advances in diagnosis and therapeutics.

1. Introduction

Trichomoniasis is an extremely common cosmopolitan non-viral genitourinary sexually transmitted infection (STI) in humans caused by the ancient protist Trichomonas vaginalis, and described decades before Chlamydia trachomatis and human papilloma virus (HPV) infection (Table 1). Worldwide 160–180 million people are affected annually by trichomoniasis. Among this population about 154 million people are in resource-limited settings, 8–10 million are in the United States and 11 million are in Europe [1]. In US the prevalence among women ranges from 2.8% (adolescents) to 51% (black communities) [2], and 51% in black men [2]. Every year a large number of asymptomatic women are attending an obstetrics and gynecology clinic. WHO has estimated that trichomoniasis accounts almost half of all curable STIs [3]. Despite these studies and high prevalence, it is one of the most poorly studied parasites with respect to virulence properties, pathogenesis and immunopathogenesis. With the development of reverse transcriptase polymerase chain reaction (RT-PCR) and nucleic acid amplification tests for T. vaginalis DNA, our understanding of the epidemiology of this pathogen has improved and suggests that as many as one-third of infections in women are asymptomatic. At present, the majority of studies are limited to adolescents and young adult women, but the prevalence of the infection has been found to increase with age. There are even fewer studies detailing its incidence and prevalence among men. More recently, acquired data provide evidence that the interactions of T. vaginalis with vaginal epithelial cells are exceedingly complex. In this review we summarize the recent series of new findings on T. vaginalis ultrastructure, pathogenesis, immunopathogenesis and new developments in diagnostics and therapeutics. Due to space limitation, we emphasize mostly recent review articles, as they include many original findings contributing to the current state of knowledge.

Table 1.

Trichomonas vaginalis (T. vaginalis) – Time line (For detail see Refs. [1,4,5,8]).

1836	• Alfred Donne identified T. vaginalis as motile microorganisms in the purulent, frothy leukorrhea of women presenting with vaginal discharge and genital irritation.
1916	• O. Hohne used the term “trichomoniasis” to describe the clinical condition of trichomoniasis, wherein T. vaginalis colonizes the vaginal mucosa.
1934–1939	• L. Procaccini identified and classified T. vaginalis as a venereal disease in a group of Italian soldiers serving in the Eastern Italian Army in Ethiopia.
1940	• R.E. Trussell found that T. vaginalis produced vaginitis in 9 of 20 women by culturing T. vaginalis.
1959	• D.H. Clark and E. Solomos developed routine culture examination for Trichomonas vaginalis
1960s–1970s	• The research focused on biochemical tests and microscopic examination to understand the growth characteristics and behavior of T. vaginalis.
1960s	• 5-Nitroimidazoles is used for the management of trichomoniasis.
1980s–2000s	• The immunologic methods and molecular biology techniques are used to study the pathogenesis and immunology of T. vaginalis.
2007	• Joint project on the T. vaginalis genome sequencing from the Institute for Genomic Research (TIGR), the Center for the Advancement of Genomics (TCAG), the J. Craig Venter Science Foundation, the Joint Technology Center and the Institute for Biology Energy Alternatives (IBEA) used whole-genome shotgun methodology (contains 1.4 million shotgun reads assembled into 17,290 scaffolds at ~7.2× coverage) to identify the genome for T. vaginalis (strain G3, ~160 megabase genome, Mbp).

2. Structure

T. vaginalis is a flagellated parasitic protozoan (protist), typically pyriform but occasionally amoeboid in shape, extracellular to genitourinary tract epithelium with a primarily anaerobic lifestyle (Fig. 1, Table 2) [4,5]. T. vaginalis is a member of the parabasilid lineage of microaerophilic eukaryotes that have unique energy-producing double membrane organelles known as hydrogenosomes [6]. Hydrogenosomes lack cytochromes, mitochondrial respiratory chain (RC) enzymes and DNA, but catalyze carbohydrate (fermentation) and produce molecular hydrogen by a diverse group of iron-only [Fe]-hydrogenases that possess a conserved H-cluster with four different sets of functional domains. T. vaginalis hydrogenosomes contain the complete machinery required for mitochondria-like organelles with FeS cluster and putative cytosolic auxiliary proteins for biogenesis of cytosolic FeS protein [7].

Fig. 1.

Morphology of T. vaginalis.

Table 2.

Trichomonas vaginalis ’s (T. vaginalis) – Key characteristics related to morphology, genetics, pathogenicity, metabolism and life cycle (For detail see Refs. [4–32]).

Shape	Amoeboid or pyriform (pear shaped)
Size	~9–23 × 7 μm (average 13 μm)
Flagella	Anterior four [9(2) + 2 arrangement] Recurrent one (originate from blepharoplast)
Internal organalles	Nucleus, Axostyle, Costa, Pelta Cytoskeleton (made up of actin, tubulin, kinesin and dynenin) Hydrogenosome without cytochrome, electron transport chain (ETC) enzymes and DNA
Genome	176,441,227 bp (strain G3), Six chromosomes ~65% Repetitive genome (includes virus-like, transposan-like, retrotransposan-like and unclassified repeats) 32.7% Guanine and cytosine (G + T) rich regions 65 Genes with short introns and conserved 12-nt sequence (5′-ACTAACACACAG-3′) at the 3′splice-site ~60,000 Protein coding genes 74 Functional core histones including 11 H2A/H2B gene pairs, 6 solitary H2A, 3 solitary H2B, 19 H3/H4 gene pairs, 2 solitary H3 and 3 solitary H4 5-Spliceosomal small nuclear RNA (snRNA-U1, U2, U4, U5 and U6) with conserved sequences and motifs similar to eukaryotes, lack 59- trimethylguanosine cap that typical of snRNAs ~250 Ribosomal DNA (rDNA) Transfer RNA (tRNAs) for all 20 amino acids ~927 Protein kinase coding genes (ePKs)
Surface proteins	~3000, Grouped into three major categories (BspA-like proteins, GP63-like proteins, adhesins and other proteins)
Energy sources	Carbohydrate via fermentative metabolism, and amino acids through arginine dihydrolase metabolism; enzyme amino-transferases and glutamate dehydrogenase synthesizes glutamate, aspartate, alanine, glutamine and glycine; enzyme cysteine synthase synthesizes cysteine; synthesize proline from arginine; synthesize phospholipids; metabolize threonine.
Adhesion proteins	Adhesion with host vaginal epithelial cells (VECs) through adhesion molecules (AP65, AP51, AP33, AP23), fibronectin binding protein, laminin binding protein and glycolipids (Lipophosphoglycan, LPG)
Life cycle	Only trophozoite stage, no cystic stage known

3. Genome and transcription machinery

T. vaginalis has a large genome (strain G3, 176,441,227 bp) with ~60,000 protein coding genes, organized into six chromosomes [7–9] (Table 2). The T. vaginalis genome contains a large number of histone genes, and most of them organize as gene pairs in a head-to-head manner. T. vaginalis has more metazoan eukaryotic transcription machinery than protistan. Introns are present in 65 genes with conserved regulatory motifs and characteristics are similar to Giardia lamblia, but not to other metazoan introns. T. vaginalis has five spliceosomal small nuclear RNAs (snRNA-U1, U2, U4, U5, and U6) without cap structure [10]. The protein coding genes in T. vaginalis are transcribed by RNA polymerase II [7]. U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III [9]. A metazoan-like TATA element is absent in trichomonad promoter, but an initiator (Inr) sequence or Inr promoter elements are present in ~75% of 5′ untranslated region (UTR) sequences of the protein-coding genes. The Inr is the only element in metazoan protein encoding genes known to be a functional analog of the TATA box for accurate transcription initiation [7]. The Inr element is located within 20 nucleotides upstream of the start codon and may be as close as 6 nucleotides. This motif with the consensus sequence TCA + 1Py(T/A), surrounds the transcription start site of all genes studied so far. T. vaginalis has an Inr-binding protein, a 39 kDa polypeptide (IBP39). IBD39 has two domains, connected by a proteolytically sensitive linker, whereas the N-terminal domain binds Inr (IBD) and the C-terminal domain (CTD) binds the RNA pol II large subunit [11]. IBD domain is a lineage-specific DNA binding domain that is utilized by specific transcription factors in T. vaginalis.

The T. vaginalis genome encodes membrane trafficking machinery, pathogenic proteins for endocytosis of host proteins and phagocytosis of bacteria and host cells that help active endocytosis and support the phagocytic life-style [7,8]. T. vaginalis has a massively amplified gene family for encoding distinct eukaryotic protein kinases (ePKs, ~880), atypical protein kinases (~40), cytosolic tyrosine kinase-like (TKL, ~124) genes without receptor serine/threonine ePKs of the TKL family, heterotrimeric guanine nucleotide–binding proteins, and components of the mitogen-activated protein kinase (MAPK) pathways, suggesting yeast-like signal transduction mechanisms. Inactive kinases are also found that make up to 17% of the T. vaginalis kinome and may act as substrates and scaffolds for assembly of signaling complexes [7,8]. ePK accessory domains are important for regulating signaling pathways. There are nine accessory domains in 8% of the T. vaginalis ePKs. The regulation of protein kinase functions and cell signaling in T. vaginalis are, however, less complex than that in higher eukaryotes [5,8].

4. Surface proteins

T. vaginalis has over 300 candidate surface proteins which belong to ten different protein families with at least one inferred transmembrane (TM) domain and share one or more features with other pathogen surface proteins. Three surface protein families are BspA-like proteins (TpLRR-containing proteins), GP63-like proteins and adhesins or others [12,13] (Table 2). The BspA-like proteins are the largest gene family encoding potential surface proteins and share a specific type of leucine-rich repeat (LRR), the TpLRR. The GP63-like proteins are the second largest gene family of candidate surface proteins encoding 77 paralogues, and 53 possess potential TM domains. GP63 are metalloproteinases (MMP) belonging to the metzincin class (EC 3.4.24.36), characterized by the motif HExxHxxGxxH (x represents any amino acid residues) forming an extended zinc-binding motif and a catalytic site. Additional candidates of this family are 28 subtilisin-like serine proteases, nine different serine proteases and five calpain-like cysteine proteases. The calpain-like cysteine proteases possess 22–23 identifiable TM domains and function as surface proteases that transport important protein fragments or small peptides for energy generation from amino acids and redox balancing. The third surface protein family of T. vaginalis shares domains with other surface proteins related to mucosal pathogens. The members of this family are identified at different stages of infection during mucosal contact and help T. vaginalis to escape from the host adaptive immune response.

5. Energy sources

T. vaginalis is a highly predatory obligate parasite that phagocytoses bacteria, vaginal epithelial cells (VECs) and erythrocytes, and is itself ingested by macrophages [5,8]. T. vaginalis uses carbohydrates as its main energy source via fermentative metabolism under aerobic and anaerobic conditions. T. vaginalis lacks the ability to synthesize many macromolecules de novo, particularly purine, pyrimidine, and many lipids, including cholesterol. These nutrients are acquired from the vaginal secretions or through phagocytosis of host and bacterial cells. T. vaginalis has a broad range of transport capabilities that facilitate transport of complex carbohydrates and amino acids through the members of the cation-chloride co-transporter (CCC) family proteins, and help survival by reflecting osmotic changes in a mucosal environment. T. vaginalis has an unusual biosynthetic pathway for synthesis of non-protein lipid anchors (inositol-phosphoceramide) of surface lipophosphoglycans (LPG) [14]. With a microaerophilic (aerobic) lifestyle, T. vaginalis uses redox and antioxidant systems to counter the detrimental effects of oxygen and express a wide range of genes encoding for defense molecules including superoxide dismutases, thioredoxin reductases, perox-iredoxins, and rubrerythrins.

6. Pathogenesis

Trophozoites of T. vaginalis are transmitted from person to person through sexual intercourse [5,16–18]. Non-sexual transmission of T. vaginalis is rare. Cystic stages are unknown for T. vaginalis. The trophozoite attaches to mucosal surfaces of the lower urogenital tract and divides by longitudinal binary fission. In vitro studies suggest that trophozoites have a 4–28 day incubation period. T. vaginalis survives long-term in the varying and adverse acidic environment of the vagina through various successful host parasitisms. VEC surface or extracellular matrix (ECM) proteins play different roles in binding diverse target molecules from the host or other microbes from the mucosal surfaces that are mediated by T. vaginalis surface proteins including LPG, cytokines, cytoskeletal protein α-actinin.

6.1. Adhesion

T. vaginalis possesses multiple mechanisms for colonization in the vaginal tract due to a dynamic hormonal influence from the menstrual cycle on the exfoliation of VECs and a constantly changing environment. After cytoadherence, T. vaginalis transforms to an amoeboid structure with increasing cell-to-cell surface contact, forming cytoplasmic projections that interdigitate with target cells. The interactions of T. vaginalis with mucin, VECs and ECM molecules persist in a non-self-limiting fashion. T. vaginalis releases cysteine proteinases into the vaginal milieu resulting in desquamation of the vaginal and cervical epithelia [19–21]. This facilitates efficient cytotoxicity toward host cells with complex interactions similar to the situation for other cell–cell contacts.⁷¹LPG is a major adherence factor in T. vaginalis, although studies of the molecular basis of adhesion of T. vaginalis to human cells have revealed that several other genes [including adhesion proteins (AP), fibronectin (FN)-binding protein, laminin-binding protein, α-actinin, enolase, phosphoglucomutase, and conserved GTP-binding protein (GTP-BP)] expressions are up-regulated [7,22–24]. At protein level four major adhesion proteins (AP65, AP51, AP33 and AP23), GAPDH and several hypothetical proteins are up-regulated in a specific receptor-ligand fashion that depends on time, temperature and pH, prompting T. vaginalis to become flat and laminate itself to the host cell [7,22–24]. Much of the evidence for the role of AP in pathogenesis has come from co-culture experiments in which antibodies to AP are shown to reduce parasite adhesion and subsequent cytopathic effects (CE) on host cells. Additionally, laminin- and FN-binding proteins are also up-regulated in T. vaginalis cytoadherence to the vaginal epithelium. T. vaginalis AP molecules bind laminin of the host target cells. FN-binding protein binds ECM components such as collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans). T. vaginalis binds to multiple FN domains including the N-terminal domain (NTD), the cell-binding domain (CBD) and the gelatin-binding domain (GBD). During these processes, iron, calcium and phosphatase are essential nutrients for differential gene expression in T. vaginalis to survive, grow, and colonize in the vaginal hostile environment.

The ap65-1 gene encodes a 65 kDa malic enzyme involved in cytoadherence. The transcription of this gene is critically regulated by the coordination of two similar but opposite oriented DNA regulatory regions, MRE-1/MRE-2r and MRE-2f; both of the regulatory proteins bind for multiple Myb-like proteins and binding varies with iron concentrations [7,25,26]. Myb1 proteins bind ap65-1 promoter at a proximal site in higher iron levels, whereas Myb2 protein binds in iron-depleted conditions [7,26]. Another iron-inducible nuclear protein (Myb3) binds only to the MRF-1 element. Moreover, Myb2 and Myb3 co-activate basal and iron-inducible ap65-1 transcription against Myb1 through conditional and competitive promoter entries. The cysteine proteinases are necessary for efficient AP-mediated adhesion of T. vaginalis to target cells. Since T. vaginalis is unable to synthesize lipids, erythrocytes may be the prime source of the fatty acids as well as iron, as an important nutrient for the trophozoids.

6.2. Hydrolases and cytotoxic molecules

A wide range of hydrolases (20–110 kDa) has been identified in T. vaginalis as cytoplasmic cysteine proteinases [5,7,8]. These hydolases have trypsin-like activity and function as cell-detaching factors by degrading proteins (such as laminin, fibronectin, and other components) of the ECM and aid in the release of host cells from tissue and mucosal desquamation. T. vaginalis proteinases of 25, 27 and 34 kDa are specifically hydrolyzed synthetic substrates with arginine–arginine residues, whereas other proteinases have activity over a wide substrate range. Four different cysteine proteinase genes of T. vaginalis have ~45% homology to cysteine proteinase genes of Dictyostelium discoideum, and are L-cathepsin and H-papain type proteinases [27]. These proteinases allow T. vaginalis to traverse the protective mucus barrier of host epithelium.

T. vaginalis produces several cytotoxic molecules and mediate cytotoxicity by damaging the target cell plasma membrane. Some of these molecules have perforin-like activity and create pores in erythrocyte membranes [5,7,8]. T. vaginalis also secretes different lytic factors (LF) with phospholipase A2 activities to destroy nucleated cells, erythrocytes and specifically degrade phosphati-dylcholine, underlying its unique pathogenesis.

6.3. Host response and innate immunity mechanism

T. vaginalis evades the immune system through complement-mediated destruction, molecular mimicry and coating itself with host plasma proteins [28–31]. Natural infection of T. vaginalis to humans seems to produce immunity that is only partially protective. T. vaginalis has unique abundant cell surface lipophosphoglycan (LPG) [28], a carbolipid molecule (2–3 × 10⁶ copies/ parasite) that similar to prokaryotic glycoconjugates. LPG plays a major pathogenic and immunoregulatory role to help adherence with VECs; triggers leukocytes to secretes interleukin-8 (IL-8), parasite-specific immunoglobulin G (IgG), IgA, Th1 cytokines, leukotrienes, reactive nitrogen intermediates and macrophage inflammatory protein-3α; induce nitric oxide synthase (iNOS); priming of helper T cells and promote transmigration of neutrophils across the endothelium [28–31]. T. vaginalis produces immuno-suppressive cytokines (IL-10, TGFβ) and causes caspase-mediated apoptosis in T-cells, macrophages and dendritic cells.

Moreover, recent comprehensive compositional and structural analysis of T. vaginalis revealed that LPG has specific LPG domains with proinflammatory properties, and its outer branch saccharide and ceramide phospho-inositol glycan core (CPI-GC) activates NFκB, ERK1/2 and MEK1/2 (28). Furthermore, T. vaginalis induces COX-2 expression, and up-regulates and activates toll-like receptors (TLR2, 4, and 9) via the p38 mitogen-activated protein kinase (MAPK) pathway. LPG CPI-GC contains terminal poly-N-acetylactosamine repeats that represent the ligand for the animal lectins called galectins [28]. Cervical and VECs release galectin-1 and galectin-3 upon T. vaginalis infection and modulate the inflammatory responses in opposite fashion (galectin-1 suppressing and galectin-3 enhancing leucocyte response to inflammatory response) [30,31]. Also galectin-1 promotes viral attachment, whereas HIV-1 infected cells enhance viral replication, gelatin-3 and cytokine expression in VECs [31,32]. Thus, natural infection with T. vaginalis results in priming of acquired immune responses. Moreover, studies from the patients infected with T. vaginalis and HIV indicated that innate immunity involves chemotaxis and subsequent influx of neutrophils [1,2,28–31].

7. Clinical signs and symptoms

Humans are the only natural host of T. vaginalis. There are two additional species of trichomonas (T. tenax, T. hominis) that infect humans but do not cause any diseases. The evidence for sexual transmission of trichomoniasis is unequivocal. Prevalence is highest among women with multiple sexual partners and with other STIs [33–35]. In women, T. vaginalis adheres to and damages VECs and causes vaginitis. Women with symptomatic trichomoniasis have a wide range of symptoms ranging from a relatively asymptomatic state to severe inflammation (Table 3). Nearly half of all women with T. vaginalis are asymptomatic. Trichomonads also affect the bladder, urethra and paraurethral glands as urinary tract infection. Moreover, women with trichomoniasis have several complications associated with adverse pregnancy outcome, preterm birth or premature labor, low birth weight, premature rupture of membranes, greater risk of tubal infertility, atypical pelvic inflammatory disease, amplified HIV transmission/acquisition, and increased risk of cervical cancer [5].

Table 3.

Trichomonas vaginalis (T. vaginalis) – Transmission, clinical signs and symptoms (For detail see Refs. [5,16,17,18]).

Transmission

Through sexual intercourse Affects genitourinary tract

Incubation period Symptoms

4–28 days In females Vaginal discharge, green to brown color (42%), frothy (~10%) Foul odor of vaginal discharge (50%) Edema or Erythema (22–37%) Pruritus Colpitis mascularis (Strawberry cervis) by punctate hemorrhages (50%) Vaginal itching Urinary tract irritation Soreness Dysuria Lower abdominal pain Elevated pH greater than 4.5, normal pH of the vagina is 3.8–4.4) Presence of amines, vaginal leucocytosis, vulvar erythema, purulent with white blood cells (WBCs) In males Symptoms of inflammation Urethral discharge Urethritis or gonococcal urethritis Irritation Non-gonococcal urethritis (NGU) or prostatitis Epididymitis Reduced sperm function Infertility

The prevalence and spectrum of trichomoniasis in males are less well characterized but the infection appears to be asymptomatic in 70% of men [5] (Table 3). In sexually active men, however, T. vaginalis causes urethritis, balanoposthitis, prostatitis, cystitis, and epididymo-orchitis and increases the risk of HIV transmission. T. vaginalis serves as a vector for the transmission of various infections. The oxidative nature of male genital fluid is, however, hypothesized to be inhibitory to certain pathogenic factors for the protozoan [5]. The presence of zinc in prostatic fluid acts as cytotoxic factor for the trichomonad. In contrast, the vagina has a reducing environment, which may contribute to the activation of some pathogenic mechanisms of T. vaginalis. Furthermore, the levels of secretory leukocyte protease inhibitor are significantly lower in T. vaginalis infected patients [5].

8. Diagnostic tests

A wide range of classic symptoms are associated with infection by T. vaginalis (Table 3), but these symptoms are similar to other STIs and cannot be used as a diagnostic markers for T. vaginalis. Thus, accurate, reliable, convenient, and inexpensive laboratory diagnostic tests play a key role in the diagnosis of T. vaginalis. These diagnostic tests are classified as old (Papanicolaou, Pap and wet mount), newer (culture and RNA), and newest (rapid antigen and nucleic acid amplification) techniques [34,36–42] (Table 4). Detailed guidelines regarding the diagnosis of trichomoniasis are available in the 2006 Sexually Transmitted Disease Treatment Guidelines published by the Centers for Disease Control and Prevention (http://www.cdc.gov/) [43]. Most commonly urine specimens are used for the diagnostic tests in male. For women, however, endocervical and vaginal swab specimens are more reliable.

Table 4.

Trichomonas vaginalis (T. vaginalis) – Diagnostic tests. In tables different T. vaginalis diagnostic tests are given for female vaginal swab and urinary samples. For males, diagnostic tests are restricted. However, for males, urinary sediments, urethral swab culture, wet mount and PCR are used. These diagnostic tests for males are ~60% sensitive and highly specific except PCR test, which is >90% sensitive and >90% specific (For detail see Refs. [34,36–43]).

	Characteristics	Sensitivity	Specificity
4A. Old diagnostic tests (Microscopic evaluation)
1. Papanicolaou (Pap) smear	Direct visualization of motile trichomonads in saline preparation Perform within 10–20 min of sample collections	50%	90%
2. Staining techniques	Can detect 3-trichomonads/ml Most common dyes are acridine orange, leishman stain, periodic acid-schiff stain, Fontana dye	30–60%	Less specific
3. Wet mount	A physiological saline preparation of vaginal secretions Trichomonads can be identified by size (similar to WBC), shape and quivering/twitching motility Require ~104 trichomonads/ml vaginal fluid	50–60%	>90%
4. The agar plate technique	A timesaving culture technique with microscopic examination of the whole clinical material obtained, require a turnaround time of 2–6 days Eliminates the need for slide preparations. Favorable for screening a low-risk population	>90%	>90%
4B. New diagnostic tests
1. Broth culture (Gold standard)	Require ~300–500 trichomonads/ml to culture at 37 °C for 2–7 days Culture media types (Diamond’s media for culture in glass tube; InPouch TV media, a double pouched soft transparent plastic container, Biomed Diagnostics, USA; Trichosel media)	~85–95%	>95–100%
2. Odor test	Whiff test (amine odor test) Perform by mixing vaginal secretions with 10% potassium hydroxide (KOH), gives strong fishy odor	–	Poor specificity due to similar results with bacterial vaginosis
3. XenoStrip-Tv technology	Antigen specific color immunochromatographic “dipstick” test (mouse antibody bound to a nitrocellulose membrane) Results can be obtained within 10 min	~66%	100%
4C. Newest diagnostic test
1. Affirm VPIII test	Unamplified RNA test (Becton Dickinson, Sparks, Maryland, USA) Rapid expensive test, require a complete laboratory, 30–60 min to perform	<90% (false +ve from dead organisms)	99%
2. Rapid antigen test	Point-of-care lateral flow test strip device (OSOM TV, Genzyme Diagnostic, Cambridge, MA, USA) Detect T. vaginalis membrane proteins within 10 min	83–90%	99–100%
3. Nucleic acid amplification test (NAAT)	Use of analyte-specific reagents to analyze transcription-mediated amplification (TMA)	96–98% (vaginal swab), 88% (urine specimen)	>98%
4. TV Polymerase chain reaction (PCR)-based test	Provides a good alternative to culture for vaginal specimens Limited availability	64–89%	97–100%

9. Nitroimidazoles

Since the 1960s, systemic treatment with 5-nitroimidazoles is the cornerstone of management of trichomoniasis. Currently metronidazole (Mz) and the other 5-nitroimidazoles (tinidazole, ornidazole, and secnidazole) have become the standard therapies for treating T. vaginalis [5,44] (Table 5). Mz is a 5-nitroimidazole, a heterocyclic compound with a nitro group on the fifth position at imidazole ring, derived from the Streptomyces antibiotic azomycin. As an inactive molecule, Mz enters T. vaginalis through passive diffusion and forms a cytotoxic nitro radical anion by anaerobic reduction through hydrogenosomes by pyruvate-ferredoxin oxidoreductase (PFOR). In this process, Mz acts as an electron sink by capturing the electrons from reduced ferredoxin, which would normally be donated to hydrogen ions to form hydrogen gas in the hydrogenase reaction. This process is induced by Mz in a concentration gradient manner in T. vaginalis. Finally, nitro radicals bind transiently to DNA, disrupt the DNA and cause cell death. Mz gel is effective in less than half the cases of trichomoniasis. Tinidazole is a second-generation nitroimidazole with a plasma elimination half-life twice that of Mz (12–14 h vs. 6–7 h). Moreover, tinidazole penetrates better into male reproductive tissues than Mz. At present, Mz and tinidazole are the only approved drugs for treatment of trichomoniasis in the USA.

Table 5.

Management of trichomoniasis (For detail see Refs. [5,44–46]).

Recommended regimens	Dose	Cure rate	Avoid medication
1. Metronidazole	2 g orally in a single dose OR 500 mg orally twice for 7 days	~90–95%.	First trimester of pregnancy and during lactation
2. Tinidazole	2 g orally in a single dose	>90%	During pregnancy, lactation or breastfeeding (should be with held ~12–24 h or 3 days after the last treatment), or in patients with blood dyscrasia or active neurological disorders

An emerging problem with Mz is rising Mz-resistance (MzR) against T. vaginalis (32.5–5% of reported cases), failure to cure the infection with two consecutive courses, and development of allergic reactions [45,46]. Resistance affects both aerobic and anaerobic mechanisms of metabolism. Resistance by aerobic mechanisms possibly arises by reduced transcription of the ferredoxin gene with decreased trafficking and activation of Mz in the cell, affecting the oxygen-scavenging pathways [5]. In anaerobic resistance, the activities of POFR and hydrogenase are decreased with concomitant reduction in hydrogen production by the impaired oxygen-scavenging mechanisms in the hydrogeno-some. Since oxygen is a highly efficient electron receptor, increased levels of cellular (hydrogenosomal) oxygen result in impaired reduction and activation of Mz. If Mz is not reduced, the concentration of the Mz is same in the intra- and extra-cellular environments, and no additional Mz enters into the cells [5]. Additionally, in the presence of oxygen, reduced Mz changes into free radical and is oxidized back to the original drug, which ultimately reduces to become superoxide anion. This process is known as futile cycling and causes only limited cell damage by the superoxide anion rather than nitro free radicals. To overcome resistance to Mz, clinicians can treat the patients with tinidazole or Mz at 2 gm orally for 10–14 days, since the ferredoxin levels and Mz resistance are inversely related.

10. Conclusions

Trichomoniasis is an extremely common infection, but it has remained ignored as a public health issue. A significant development has taken place in diagnosis and therapeutics to resolve trichomoniasis. Current treatment with nitroimidazoles is reliable and inexpensive, but the number of resistant strains is increasing. Moreover, nitroimidazoles treatments are not recommended for pregnant and lactating women due to incomplete establishment of safety issues. Therefore, more studies are needed to determine the most sensitive diagnostic kits with high specificity associated with rapid read-outs as well as to expand therapeutics in both men and women.

Abbreviations

Ad

adhesion molecules

AT

agglutination test

CCC

cation-chloride cotransporter

CBD

cell-binding domain

CDC

Centers for Disease Control and Prevention

CE

cytopathic effects

ePKs

eukaryotic protein kinases

ECM

extracellular matrix

Fdx

ferredoxin

FN

fibronectin

GBD

gelatin-binding domain

GPI

glycosylated phosphatidylinositol

GU

gonococcal urethritis

HIV

human immunodeficiency virus

HPV

human papillomavirus

IL-8

interleukin 8

LRR

leucine-rich repeat

LPG

lipophosphoglycan

LF

lytic factors

Mz

metronidazole

MzR

metronidazole-resistant

MAPK

mitogen-activated protein kinase

NTD

N-terminal domain

NGU

non-gonococcal urethritis

NAAT

nucleic acid amplification test

Pap

Papanicolaou

PE

phospholipids phosphatidylethanolamine

PCR

polymerase chain reaction

KOH

potassium hydroxide

NADPH

reduced nicotinamide adenine dinucleotide phosphate

STI

sexually transmitted infections

snRNA

small nuclear RNAs

TMA

transcription-mediated amplification

tRNAs

transfer RNA

TM

transmembrane

TKL

tyrosine kinase-like

UTR

untranslated region

UTI

urinary tract infection

VECs

vaginal epithelial cells

VSP

variant surface proteins