INTRODUCTION

Vaginal discharge is the most common cause of gynecological consultation ¹. The most common vaginal discharges are bacterial vaginosis (BV) produced by Gardnerella vaginalis (Gv), Candida albicans (CA) and Trichomonas vaginalis (Tv) ^2,3. Table I shows the most common causes of vaginal discharge ⁴. Tv is the most common, prevalent, curable non-viral sexually transmitted infection (STI) worldwide ^5,6.

METHODOLOGY

Literature searches were performed electronically in PubMed, Medline, ISI, Springer, Embase, Web of Knowledge, DOAJ, Google Scholar and the Cochrane Library for original articles written in the English language and in Scielo, Latindex, Imbiomed- L, Redalyc and Google Scholar for original articles written in the Spanish language. Selection criteria included randomized clinical trials, observational trials, open-label non-randomized trials, and case reports related to Tv and cervical cancer. The Cochrane Library was searched for reviews. Publications from 1970 to June 2020 were reviewed. Three hundred and ninety references were found and 150 met the inclusion criteria.

The searches included the key words (Mesh): Trichomonas vaginalis, epidemiology of Trichomonas vaginalis, epidemiology of cervical cancer, Trichomonas vaginalis and infection, inflammation mechanisms, Trichomonas vaginalis and inflammation mechanisms, Trichomonas viruses, carcino genesis, cervical cancer and co-factors, sexual transmitted infections and cervical cancer, cancer and inflammation mechanisms, Trichomonas vaginalis and cervical cancer. The electronic search and eligibility of the studies were evaluated by the author. The author reviewed, analyzed and discussed the Trichomonas and its role in cervical cancer.

Epidemiology

According to World Health Organization (WHO) ², there are more than one million curable STIs that occur each day. Globally, it is estimated that for 2016, there were roughly 376 million new infections of the four curable STIs: Clamydia tracomatis (Ct), gonorrhea, syphilis and trichomoniasis as seen on Table II ^2,6. According to different authors ^2,6 the prevalence of Tv for 2016 was estimated in 5.3% (95% IC: 4.0- 7.2) in women and 0.6% (95% IC: 0.4-0.9) in men, with regional values ranging from 1.6 to 11.7% in women and from 0.2 to 1.3% in men. Rowley et al⁶ reported a Trichomoniasis incidence of 40 per 1000 in women (95% IC: 27-58) and 42 per 1000 men (95% IC: 23-69). Although Tv is a worldwide parasite, prevalence and incidence rates differ or depend on the part of the world, the culture, the religion and the population studied ^5,7. Tv is considered as the second most frequent STIs and the second most common cause of lower genital tract infection worldwide ⁸. WHO ² reported that the African Region had the highest incidence rates for trichomoniasis in women and men. In the America region, the Pan-American Health Organization ⁹ reported an incidence of 18.8 million new cases of trichomoniasis among females, and 13.6 million new cases among men and a prevalence of 18.8 million existing cases of trichomoniasis among females, and 3.2 million existing cases among men in 2012. According to the Center for Disease Control and Prevention ¹⁰, the prevalence in USA of trichomoniasis is 2.1% in women between 15-49 years old and they estimate that 3.7 million people have trichomoniasis. Studies in Latin America revealed prevalence values of 7.6% in Argentina ¹¹, 7.8% in Chile ¹² and 9.1% in Peru ¹³. In Brazil, prevalence ranged from 2.6% to 20% among women ^14-16. In Venezuela, Núñez et al¹⁷ reported a prevalence of 7%. The incidence of Tv infection depends on several risk factors including age, sexual activity, number of sexual partners, sexual partner promiscuity, menstrual phase, prostitution, imprisionment, use of illicit drugs, race, diagnostic techniques, low social and economic conditions, as well as other STIs such as Herpes Virus 1 and 2(HVS), Ct, CA, Gv, Gonorrhea (G), Human Papilloma Virus (HPV), Human Immunodeficiency Virus (HIV) ^10,18. The prevalence is high among low social income patients from gynecologic and STI clinics ¹⁸. Patel et al. ¹⁹ using urine samples found that Tv infection prevalence was 0.5% and 1.8% among males and females, respectively. Tv infection prevalence was 4.2% among black males, 8.9% among black females, and 0.03% and 0.8%, respectively, among males and females of other races/ethnicities. Tv infection prevalence was positively associated with female sex (OR: 6.1; confidence interval 95%(IC):3.3- 11.3); black race (vs other races/ethnicities OR:7.9 IC 95%:3.9-16.1); older age (vs 18- 24 years, OR: 3.0 IC 95%:1.2-7.1) for 25- to 39-year-olds and OR: 3.5, IC 95%: 1.3-9.4) for 40 to 59-year-olds); having less than a high school education (vs completing high school or more, OR: 2.0, IC 95%:1.0-4.1); being below the poverty level (vs at or above the poverty level, OR: 4.0, IC 95%:2.1-7.7); and having ≥2 sexual partners in the past year (vs 0-1 sexual partners, OR:3.6, IC 95%: 2.0-6.6).

Tv infection occurs in the female and male urogenital tract and humans are the only natural host for the parasite ^18,20. As all STIs, Tv is transmitted by sexual intercourse and the evidences that corroborate for the classification of trichomoniasis as STI are: 1.- high frequency of infection in urethra and/or prostate of male partners of infected women; 2.- the prevalence of infection is higher among female attending in STD clinics and among prostitutes than in postmenopausal women and virgins; and 3.- Tv die outside of the human body, unless they are protected from desiccation ^18,21. Tv infection results in a variety of clinical manifestations in most cases the patients are asymptomatic, but some may develop signs typically associated to the disease. Moreover, beyond the symptoms, the main issue concerning trichomoniasis is its relationship with serious health consequences such as cancer ^22,23, adverse pregnancy outcomes ^24,25, infertility ^26,27, and HIV transmission and acquisition ²⁸. The infection was traditionally known as symptomatic in women and asymptomatic in men ¹⁸, however, this currently is changing and recent data have mentioned that around 80% of Tv infections are asymptomatic in both men and women ²⁹. Symptomatic women ranged between 50-85% ^21,30-32 while in men the percentage was 15-77% ^30,31.

Pathobiology/Pathophysiology

Tv was discovered in 1896 by the French medical doctor and microbiologist, Alfred F. Donné; in 1916, Hoehne showed the Tv was responsable for producing vaginitis ³³. Tv belongs to the Trichomonadidae family. Four different types of Trichomonas (T) can be found in humans: T. hominis, T. tenax, T. vaginalis y Pentatricomonas hominis (Ptv). The T. hominis is found in the intestines, the T. tenax in the mouth, the Ptv in the intestines and Tv in the vagina and uretra. As it was mentioned, they parasite the urogenital area of women and men ⁵. Tv is a flagellated parasitic protozoan, typically pyriform but occasionally amoeboid in shape, extracellular to genitourinary track epithelium with a primarily anaerobic lifestyle ³⁴. The individual organism is 8-20 µm long and 2-14 µm wide. Four flagella project from the anterior portion of the cell and one flagellum extends backwards to the middle of the organism, forming an undulating membrane. Tv has a foamy, cyanophilous cytoplasm (greenish grey on a Pap smear) with red granules, and eccentric, possibly red-coloured nucleus. An axostyle extends from the posterior aspect of the organism. Parallel to this membrane, a bundle of microtubules called the costa, is arranged inside the cell. It has a Golgi apparatus associated with microfilaments (the parabasal filaments) that, together, form the so-called parabasal body. Traversing the cytoplasm as an axis and protruding from the posterior end, it presents a structure also made up of microtubules called the axostil. This axostil, in its anterior part, widens and partially covers the nucleus. As a continuation of the axostil towards the anterior part, there is another microtubule structure, the pelta, which partially covers the basal structures of the flagella. The nucleus, which has an endosome, is arranged in the anterior zone, near the point of insertion of the flagella. In stains, the parabasal nucleus-body-axostil (anterior part) flat assembly is usually stained as a single mass. It lacks mitochondria and instead has organelles called paracostal bodies (because it is close to the costa) and paraxostillary bodies (because it is close to the axostil), which are hydrogenosomes, whose function is to produce energy (ATP) under anaerobic conditions ⁵. This hydrogenosome, is a mitochondrion-like organelle that generates hydrogen ³⁵. Due to its microaerophilic lifestyle, Tv does not have the ability to generate ATP by oxidative phosphorylation but depends on substrate level phosphorylation. Originally, it was assumed that the hydrogenosome is an ancestral form of the mitochondrion. It is now apparent, based on phylogenetic studies, that the hydrogenosome constitutes a reduced form of fully developed mitochondria. Nevertheless, the hydrogenosome remains an intriguing organelle, and the extraordinary size of the Tv genome, exceeding 160 Mb, will certainly provoke further research in the years to come ^36,37. Tv has a large genome (strain G3, 176,441,227 bp) with ~60,000 protein coding genes organized into six chromosomes ³⁸. Tv is a highly predatory obligate parasite that phagocytoses bacteria, vaginal epithelial cells and erythrocytes, and is itself ingested by macrophages. Tv uses carbohydrates as its main energy source via fermentative metabolism under aerobic and anaerobic conditions ^31,32.

Its incubation time is generally between 4 and 28 days ²¹. Tv primarily infects the squamous epithelium of the female and male urogenital tract. Tv resides in the female lower urogenital tract (vagina, urethra) and the male urethra and prostate, where it replicates by binary fission. Tv is transmitted among humans, its only known host, primarily by sexual intercourse. Infection may persist for long periods, possibly months or even years, in women but generally persists less than 10 days in males ³⁹. The parasite does not appear to have a cyst form and does not survive well in the external environment, but can survive outside the human body in a wet environment for more than three hours ^5,31. However, there may be a pseudocyst form. Tv pseudocyst has been found to be more virulent in animals and could have relevance for humans, particularly in the case of cervical neoplasia ^40,41. Tv can be infected with double-stranded RNA (dsRNA) viruses that may have important implication for trichomonal virulence and disease pathogenesis ^41-43. As it was mentioned before, Tv is transmitted during sexual intercourse; men also become infected (asymptomatic hosts), but most commonly exhibit urethral infections. Tv has to deal with constant stress in its physiological niche of the urogenital tract that includes changes in pH, fluctuation in iron balance and other nutrients and desquamation of vaginal epithelial cells associated with menstrual cycle ⁴⁴. In addition, the parasite may reside in the urethra of women from where it reinfects the cervico-vaginal segment upon the pH increase. When the lactobacilli have reinstated, the vaginal pH level returns to normal and the parasite disappears from the area since it cannot survive in an acid environment. Hence, the conclusion of some specialists according to which, some women heal by themselves. In fact, the parasite present in the urethra reinvades the vagina in the period prior to menses, when the pH increases to 7.5 and after the menstrual cycle, when the pH decreases again to 4.0-4.5, Tv moves back into the urethra. Thus, in the absence of a sustained therapy, reinfection occurs. The parasite adheres to the wall of the cervicovaginal cells and, by a very effective enzyme system, it interferes in the metabolism of the host generating toxins that may lead to cell necrosis. In addition, for survival, the parasite needs very little energy, which it acquires by the glycolysis of cellular glycogen ^5,45. It is known that parasitic protozoans have a variety of complex carbohydrates on their surfaces, e.g., glycolipids, glycoproteins, and lipid-anchored glycosylated phosphatidylinositol (GPI). These glycoconjugates have been reported to play important roles in host cell invasion and evasion of host immune responses by several protozoa ^46,47. Fichorova et al. ⁴⁸ have demonstrated that Tv express lipophosphoglycans (LPGs) (2 106 to 3 106 copies/ parasite) anchored on the cell surface via an inositolphosphoceramide ^49,50.

The high density of lipophosphoglycans (LPGs) on the parasite surface suggests that they have important roles in parasite biology and in the infections they cause. The same authors ^49,50 previously showed that pretreatment of trichomonads with periodate abolished adhesion of specific parasites to their respective host cells, implying the involvement of carbohydrate molecules in these processes ⁵¹. This is of a fundamental importance to understand the pathobiology of trichomoniasis and to define the role that this molecular component(s) play(s) in the infection of host tissues, in particular vaginal epithelial cells (VECs). Fichorova et al. ⁴⁸ confirmed the role of surface carbohydrates in the adhesion of Tv to host epithelial cells in the lower female genital tract and strengthen the evidence of LPG involvement in this process.

Adhesion

In order to survive as an extracellular parasite, Tv must adhere to the epithelial lining or extracellular matrix components of the urogenital tract especially to VECs ^52,53. Attachment to cells, microorganisms or other surfaces, drives a transition of the ovoid free-swimming parasite into an amoeboid form which it is highly adherent ⁵³ (Fig. 1). In addition to VECs, Tv can bind diverse cellular and non-cellular structures upon which a similar transition to an amoeboid form is observed. This suggests that the parasite utilizes either an adaptable non-specific or multiple specific binding mechanisms to mediate attachment and signal its morphological remodelling ⁵².

Fig. 1 T. vaginalis interaction with extracellular membrane (ECM) and host cell plasma membrane (PM). The ovoid free-swimming form of Trichomonas (upper left-hand side) transitions to an amoeboid form upon binding to a host epithelial cell (upper right-hand side). Lower panel: various T. vaginalis membrane molecules: LPG, hypothetical proteins (Hyp Prot), membrane proteins with conserved domains (Cons Memb Prot) and dual-functional proteins (Dual Fxn Prot) implicated in the interaction with ECM and host cell PM receptors. Galectin-1 (Gal-1) is the only reported host cell receptor. Adapted from: https://www.google.com/search?q=Trichomonas+vaginalis:+current+understanding+of+host%E2%80%93parasite+ interactions&sxsrf=ALeKk01yqd1vojQZy74KRaq3mf_VB1SNVA:1596481107780&source= lnms&tbm=isch&sa=X&ved=2ahUKEwjpgsfK2_qAhUB66QKHUfaDoIQ_AUoAXoECA4QAw&biw= 1440&bih=701#imgrc=u0vsGUaBogCy0M. 

Multiple molecules have been postulated to be involved in attachment of T. vaginalis to target cells (Fig. 1). These adhesion molecules can be separated into three categories: 1. a controversial set of metabolic proteins that appear to have secondary adhesive properties; 2. a lipophosphoglycan; and 3. a collection of membrane proteins, many of which have been recently identified through genomics and proteomics ⁵².

Metabolic proteins

The class of proteins designated as adhesion proteins (APs) consisting of AP23, AP33, AP51, AP65 and AP120 are the most controversial of the adhesion molecules ⁵⁴. However, it has also been reported that these proteins are present on the surface of the parasite ^55,56. Finding proteins with multiple functions and localizations is reasonable; Alderete et al.⁵⁷ have reviewed about this dual function of these enzymes as metabolic proteins and adhesins. Other research groups, however, have provided immunofluorescent microscopic evidence that these molecules are located exclusively in the hydrogenosome, denying the dual localization ⁵⁴. It has been shown that AP23, AP33, AP51 and AP65 adhere to several cell types and bind to these targets in the absence of membrane proteins ^54,58. These results suggest a non-specific membrane binding, a property that would argue against a precise role in pathogenesis. Recent research shows that AP51 and AP65 bind to hem and hemoglobin, a property that paradoxically supports their promiscuous nature and lack of adherence specificity and implies a role in binding to a key nutrient ⁵⁹.

It has recently been proposed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, is involved in adherence of Tv to the host extracellular matrix protein fibronectin and is considered with dual-functional: adhesins/enzymes ⁶⁰.

Lipophosphoglycan

Tv is coated by a complex structure of glycoconjugates known as a glycocalyx. Studies of this glycocalyx began almost 40 years ago with looking lectin at the parasite surface ⁶¹. This investigation has been narrowed to a single surface glycan: LPG. Studies have suggested that surface carbohydrates, specifically terminal N-acetylgalactosamine or galactose, are important in determining the virulence of Tv. It was found that treating parasites with glycosylases caused a more than 20-fold reduction in binding of parasites to human VECs ⁶². In addition, exposure of Tv to periodate to oxidize the glycocalyx reduced parasite binding to laminin and adhesion to human VECs ⁵¹. The first, and only, surface glyconjugate isolated from Tv is the most abundant. With 2-3×10⁶ molecules per cell, Tv LPG was thought to be a probable candidate to play a role in host-parasite interactions ⁴⁹. This complex molecule is composed of an inositol-phosphoceramide anchor ⁵⁰ and a polysaccharide core composed of glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, rhamnose and xylose at a composition of 4.2%, 14.6%, 35.8%, 2.4%, 27.1%, 15.9%, respectively. Several important biological functions have been attributed to Tv LPG. Several lines of evidence demonstrate that Tv LPG is an adhesion molecule: 1. when LPG is added, the Tv biding to the epithelial cells are inhibited to epithelial cells ^48,49,63; 2. LPG binds to human galectin-1 and this interaction results in host cell attachment ⁶⁴. Although it is unclear whether galectin-1 binding causes alteration in host cell gene expression, LPG exposure to several epithelial cell types up-regulates interleukin-8 and macrophage inflammatory protein 3a expression ⁴⁸. Furthermore, LPG activates the pro-inflammatory transcription factor nuclear factor κB ⁶⁵. These activities support a role for LPG in parasite attachment, specificity of binding and manipulation of host cell-gene expression. Even though several functions of Tv LPG have been defined, important questions remain unanswered. It is unclear if galectin-1 is the only receptor for LPG, as several other galectins could function as receptors, particularly galectin-3, which is expressed by VGEs ⁴⁸.

Membrane proteins

When attempting to identify proteins involved in the attachment of a parasite to a host cell, an obvious starting place is proteins anchored to the parasite's surface. Several observations suggest that Trichomonas surface proteins are involved in attachment to host cells and structures. Trypsinization of the surface of Tv decreases binding of the parasite to laminin, fibronectin and several human cell lines ⁶⁶. It has been found a membrane-localized protein is involved in the attachment of Tv⁶⁷. The completion of the Tv genome ³⁸ has launched investigations of protein families that contain transmembrane domains and domains shared with surface proteins implicated in pathogenesis in other organisms ⁵⁴. Four families encompassing 128 genes, encoding serine, cysteine and metalloproteases were identified. These proteases may play a role in degrading host proteins or extracellular matrix to clear attachment sites ⁵⁴. Alternatively, they may be involved in degrading parasite-host bonds allowing the parasite to be released and transmitted to the next host. Additionally, three gene families encoding 47 proteins similar to surface proteins expressed in other mucosal pathogens, chlamydial polymorphic membrane proteins, immunodominant variable surface antigens and giardial VSP (variant surface protein)-like proteins, were identified ⁵⁴. These bioinformatic analyses also revealed the presence of genes encoding 11 surface lectins that may bind to carbohydrates in the extracellular matrix or on the host cell ⁵⁴. This possibility is strengthened by a previous observation that treatment of host cells with periodate to oxidize carbohydrates reduced parasite binding ⁶⁸. Finally, the largest family identified through these studies was the BspA-like group named for their similarity to leucine-rich proteins of mucosal bacteria known to mediate adherence to host cells ⁵⁴. This gene family was initially found to encode 658 proteins, but further analysis revealed 911 proteins with 191 containing predicted transmembrane. Furthermore, indirect immunofluorescence shows that at least one family member, TvBspA-625 (where TvBspA is Tv BspA), is expressed on the surface of the parasite ^54,69. Future experiments are needed to determine the expression patterns of predicted membrane proteins and to provide direct evidence for their involvement in the interaction of Tv with its host. Understanding both the function and expression patterns of these proteins will be critical to determine whether any, may be valid targets for rational drug or vaccine design.

Virulence

The importance of maintaining an intact mucosal layer is crucial to avoid the occurrence of pathological disorders associated with inflammatory disease, in which disruption of the epithelial barrier leads to severe inflammation of the mucosal tissue compartments ^70-73. The epithelial barrier is maintained by connections between adjoining epithelial cells, which are the first sites of contact with the host. These cells are responsible for separating potentially harmful luminal content from the underlying tissue. This function of acting as a physical barrier is accomplished by junctional complex, which comprises a plethora of membrane-associated and transmembraneous proteins organized in discreet, spatially restricted complexes ^72,73. For establishing infection, Lin et al. ⁷⁰ findings provide solid evidence to support claims that the damages of Tv infection to host cells and cell junctions is initiated by the attachment of live parasites. Tv requires to break through the mucus layer and adhere to epithelial cells of urogenital tract ⁷⁴. It also penetrates into basement membrane and binds to extracellular matrix proteins ⁷⁵. Previous reports have shown that host cells infected by Tv presented disruption before being lysed ^76,77.

Epithelial cells act in a manner similar to that of a connector in a host-microbe communications network, building signal transduction connections between luminal microbes and the host ^76,77. Tv virulence depends on two factors: cytoadherence and cytotoxicity ⁷⁸. Studies using the scanning electronic microscope and transmission electronic microscope on samples collected from patients with Tv vaginitis showed that the capacity to adhere to VECs lead Tv to development of pseudopodial and filopodial processes giving a distinct form which is spherical or ovoid ^79-82. Furthermore, there is a reason to suspect that the adhesiveness and amoeboid form of Tv contribute in an important way to the pathogenesis and virulence. Health ⁸¹ found that the damage to VEGs is related to the adhesion of this amoeboid form to the cells. The adhesiveness is a mechanism for increasing the efficiency of parasite-derived cytotoxic and cytolytic factors since they could act at short range between the VEC and the adhering amoeboid Tv⁸¹. Also, several products from the parasite's metabolism such as lactic acid, hydrolytic enzymes: hyalurodinase, glicosidases, mucinases, adhesins, and acid phosphatase contribute to the cytotoxitciy ⁷⁴. The amoeboid movements of Tv, acting alone or together with the sectored factors, and provide a mechanism for injuring the epithelial cells. Heath ⁸¹ showed that this amoeboid movements of Tv are important in disrupting the epithelial cells. The author ⁸¹ concluded that both mechanisms, mechanical and chemical, work in the human Tv infection. The adhesion of Tv to the vaginal and exocervical epithelia could be an important role in disrupting the superficial layers of squamous epithelium causing the necrosis and increasing the rate of desquamation which are very common features of severe Tv vaginitis. Also, some authors have mentioned that there is an increased in the sub-epithelial vascularity of the vagina and cervical walls, in other words an acute inflammation ^80,82. Trichomonas requires to break through the mucus layer and adhere to epithelial cells of the urogenital tract ⁷⁴. It also penetrates into the basement membrane and binds to extracellular matrix proteins ⁷⁵. Although pathogenesis and virulence in human trichomoniasis is not fully understood, progress has been made in identifying parasite products that can damage host cells and tissues.

Molecular mechanisms

Adhesion is thought to play an important role in the pathogenesis of trichomoniasis, and investigations of the molecular basis of adhesion of Tv to human cells have identified several adhesion molecules (Ad) on the surface of the parasite. Much of the evidence for the role of Ad in pathogenesis has come from co-culture experiments in which antibodies to Ad were shown to reduce parasite adhesion and subsequent cytopathic effects [CE] on host cells ⁸³. Additional studies had shown that contact of Tv with mammalian targets caused up-regulation of Ad and that the parasite assumed a flattened shape, essentially laminating itself to the host cell ⁷⁵. Cysteine proteinases seem to be necessary for efficient Ad-mediated adhesion of parasites to targets ⁸⁴. Control of the expression of Ad also seems to be under the influence of iron, since transcription induction of ap65-1 (adhesion protein 65,000) was reported to be regulated, via certain iron-responsive DNA elements (e.g., AGATAACGA) ⁸⁵. The Ad family appears to be regulated at several levels. These findings suggest that adhesion of Tv facilitates efficient cytotoxicity toward mammalian cells and probably involves complex interactions similar to the situation for other cell-cell contacts such as leukocytes ⁸⁶. Additional parasite molecules with functional carbohydrate groups, distinct from Ad, appear to be involved in the adhesion of Tv to VEC ⁸⁶, although the precise roles of the molecules in parasite adhesion and CE are not known. Little is known about the host cell receptors to which parasite adhesion molecules bind; although there is some evidence that laminin may be a target for trichomonad adhesion ⁸⁷.

Hydrolases

A variety of hydrolases have been described in Tv, with cysteine proteinases being particularly prevalent. Ranging from 20 to 110 kDa, several of these lower molecular mass proteinases are released from the Trichomonads ^88-90. There has been described other Tv products such as cell-detaching factors which are released by the parasite ^91,92; it is known that some of them do have trypsin-like activity. These factors are active on human cells, causing them to detach. The release of cell-detaching factors and proteinases from Tv clearly implies that these parasite products could degrade proteins such as laminin, vitronectin, and other components of the extracellular matrix, thus effecting the release of host cells from tissue. In addition, the levels of secretory leukocyte proteases inhibitor in patients with Tv infections are significantly lower than those in uninfected patients ^93,94, The substrate specificity and structure of some of the proteinases of Tv are now being determined ⁸⁸. Proteinases with lower molecular masses are released from the parasite; proteinases of 25, 27 and 34 kDa specifically hydrolyzed synthetic substrates with arginine-arginine residues, whereas other proteinases have activity over a wide substrate range ^88,90.

Cytotoxic molecules

Evidence suggests that Tv may produce molecules that are delivered to target cells and mediate cytotoxicity through damage of the target cell plasma membrane. One of these molecules creates pores in erythrocyte membranes as detected by electron microscopy ⁹⁵, thus displaying perforin-like activity ⁹⁶. An additional membrane-attacking molecule has recently been detected in Tv, a lytic factor (LF) is released by Tv that can destroy nucleated cells and erythrocytes and specifically degrades phosphatidylcholine, suggesting that it is a phospholipase A2 ⁹⁷.

Other molecules

The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in trichomonads, was confirmed by showing that the parasite responds to environmental changes, resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels ⁸⁸.

Viruses

Another factor that could increase its virulence is the presence of concomitant viruses ⁹⁸. In 1985 it was first observed that a virus-like particles infected the protozoans, and were later recognized as being components of a double-stranded RNA (dsRNA) virus infecting the protozoans ^99-101. Tv virus (TvV) is a non-segmented, 4.5-5.5 kilo-base pair (kbp), They are a dsRNA virus belonging to the genus Trichomonaviruses and to the viral family Totiviridae¹⁰². TvV is encased within an 85 kilodalton major viral protein capsid arranged in the shape of a 120-subunit icosahedral and is closely associated with the Golgi complex ¹⁰³. TvVs use a viral RNA-dependent RNA polymerase to replicate its genetic material. TvVs affect the total protein expression of Tv, and especially the expression of cysteine proteinases and the immunogenic protein P270, which are linked to cytotoxicity, cytoadherence, degradation of basement membrane of epithelium and host immune evasion ^{78,98,104,105}. TvVs can be divided into four different viral strains that have the ability to co-infect Tv at the same time: TvV1, TvV2, TvV3, and TvV4 ¹⁰⁶. Each TvV strain has different effects on various aspects of Tv pathogenesis. TvV1 and TVv2 have been linked to genital symptom severity, while TvV2 and TvV3 are involved in the up-regulation of the surface expression of P270, cysteine proteinases and other virulence factors of Tv that help the Tv evasion from the host's immune response. The role of TvV4 has yet to be elucidated ^98,106,107. However, TvVs are considered to be well adapted to Tv such that they cause few if any deleterious effects in the protozoan and maintain a largely non-cytopathic and persistent infection ¹⁰².

Despite their non-lytic life style, infection of Tv by the dsRNA viruses causes the up-regulation of synthesis and surface expression of a highly immunogenic protein, P270 ^104,108. The presence of dsRNA viral infection of Tv is also associated with differential qualitative and quantitative expression of cysteine proteinases ⁷⁸. Cysteine proteinases are linked with Tv cyto-adherence to vaginal epithelium, cytotoxicity, and degradation of basement membrane components (109). Two Tvs surface proteinases bind to host epithelial cells and are related to levels of cytoadherence and cytotoxicity ^109,110. Thus, dsRNA viruses induce various phenotypic changes that may impact Tv virulence ⁷⁸. Type 1 is the termed used to name virus negative Tv and type 2 for Tv positive to virus ¹⁰⁸. The biological significance of Tv infection by TvVs remains poorly understood. It appears that TvV infection is generally a noncytopathic phenomenon that leads to stable, persistent infections of the protozoan host without an extra-cellular transmission phase ¹¹¹. Transmission of TvV occurs during cell division and possibly also during mating ¹¹². Infection of Tv by TvVs could modulate the pathogenicity and virulence of Tv infections of the human genital mucosa ¹⁰⁶. The percentage of TvVs-infected-Tvs ranged from a low of 30% ¹⁰⁰ to a high of 100% ¹¹³. Graves et al.⁹⁹ reported a 40% of Tvs infected by TvVs. Wendel et al. ¹⁰⁸ and Graves et al. ⁹⁹ found that the Tvs infected by TvVs were older (median age 38 years). These authors ^99,108 hypothesized that older women with Tv have less immune surveillance against TvV. However, Heidary et al. ¹¹⁴ did not find an association between TvV+ isolates and older age. Thus, the relationship between TvV positivity and older age remains controversial, and additional studies are needed.

Inflammation

Epithelial cells act as a connector in a host-microbe communications network, building signal transduction connections between luminal microbes and the host. The epithelial cells act as sensors for the microbes and as providers of signal to immune cells to activate inflammatory and immune responses ¹¹⁵. Twu et al.¹¹⁶ demonstrate that Tv exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. In addition, they showed that ¹¹⁶ parasite exosomes act to modulate host:parasite interactions and promote exosomes in promoting parasite:parasite communication and host cell colonization. Lin et al.¹¹⁵ showed that there are seven cytokines expressed in the Tv infection; among them, is IL-8. A few days after the human become infected by Tv, there is a degeneration of the vaginal epithelium, followed by leukocytic neutrophil infiltration that is associated with abundant vaginal discharge and intensely inflamed vaginal tissues. However, little is known about the early mechanism of how neutrophils accumulate or how epithelial cells mediate the initial inflammatory response upon Tv infection. Fichorova et al. ⁴⁸ have provided evidence for an early involvement of chemokine production by epithelial cells that occurs in response to nontoxic doses of LPG and precedes cytotoxic effects. Interleukine-8 (IL- 8) is known to govern polymorphonuclear leukocytes localization and function ¹¹⁵ and is the major chemokine responsible for neutrophil recruitment to sites of tissue insult and inflammation ^117-119, Shaio et al. ^120,121 reported the interleukin-8 response to infection with Tv in human monocytes and neutrophils. Also, the same authors ^120,121 found the presence of IL-8 in the vaginal discharge from patients with symptomatic trichomoniasis, providing evidence for involvement of IL-8 in the inflammatory response to Tv. Production of IL-8 by monocytes and neutrophils stimulated by live trophozoites ¹²² or undefined Tv fragments has been reported ¹²¹. They ^121,122 have reported species-specific IL-8 production by human vaginal, ectocervical, and endocervical epithelial cells, in response to a chemically defined T. vaginalis constituent, purified T. vaginalis LPG. Increased IL-8 levels in genital tract secretions and amniotic fluid have been correlated with upper genital tract infections and preterm labor ¹²³, suggesting that Tv IL-8 up-regulation may underlie the mechanisms linking trichomoniasis with preterm labor.

It has been hypothesized that the production of IL-8 during Tv infection may be secondary to the cytopathic effects and the release of the early response pro-inflammatory cytokines: Tumor Necrosis Factor-alfa (TNF-α) and Interleukine-1beta (IL-1β), by damaged epithelial cells. Fichirova et al.⁴⁸ demonstrated that IL-8 is secreted by the cervical and vaginal epithelial cells, with no storage of significant IL-8 amounts in the epithelial cytoplasm. On the other hand, large intracellular stores of IL-1 and some small amounts of TNF-α are present in these cells and released once the membrane rupture and cell death ⁴⁸. TNF-α and IL-1β induce the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation via TNF receptor I and IL-1 receptor I, respectively, and also may act as toll-like receptor agonists in the reproductive tract epithelial cells ^119,124. The notion of the primary role of IL-1β and TNF-α in the Tv-induced inflammatory response has found indirect support in the evidence of increased cervico-vaginal levels of these cytokines under pathological conditions commonly associated with trichomoniasis, e.g., vaginitis, bacterial vaginosis, HIV-1 infection, and preterm labor ¹²⁵. However, Fichirova et al.⁴⁸ showed that LPG stimulates a significantly increased IL-8 production in the absence of cell toxicity and at low baseline levels of endogenous IL-1β and TNF-α. These findings suggest a direct recognition rather than a secondary cytopathic or host factor-mediated signaling effect of Tv LPG and support the hypothesis that the chemokine response to Tv may be initiated via LPG interaction with VEC surface receptors. Although these results indicate that LPG can trigger responses directly in VECs, and that this effect does not discard LPG interaction with leukocytes, such as macrophages, during the subsequent inflammatory response that may occur by other mechanisms and have a potent contribution to the pathogenesis of trichomoniasis. Fichorova et al.⁴⁸ mentioned that the MIP-3 production could play an important role in the immune response to Tv infection in the VECs. MIP-3a is known to recruit Langerhans-like CD34 progenitor dendritic cells (DCs) to the site of inflammation, by guiding them as they traverse the lamina propia on their way to the epithelial surface, a journey which precedes their maturation ¹²⁶. Also, Fichorova et al.⁴⁸ found that LPG-induced IL-8 and macrophage inflammatory protein-3 (MIP-3) a production by the VECs is, at least, partially MyD88 independent. This cytoplasmic adaptor protein MyD88 is essential for rapid NF-kB activation and cytokine production in response to multiple TLR ligands including LPS ¹²⁷. The MyD88-independent activation via TLR is important for DC maturation ¹²⁸. Fichorova et al. ⁴⁸ have presented evidences of the major role of Tv LPG in the pathogenesis of the mucosal inflammatory reaction and suggested its modulatory role in the innate host immune response through selective species-specific cytokine up-regulation and multiple signaling pathway. Lin et al.¹¹⁵ found that the vascular endothelial growth factor Beta (VEGF-β) is induced in human vaginal and ectocervical cells infected with Tv. According to these results, it is reasonable to assume that the disruption of host cell pathogenesis may be caused by these cytokines. Another cytokine which increased expression level, BMP-2 is a member of the TGF-β superfamily, playing an important role in the embryonic development, suppression of the immune response, and differentiation and proliferation of tissues and cells. Fichorova et al⁴⁸ have provided evidence that the major role of Tv LPG in the pathogenesis of the mucosal inflammatory reaction and suggests its modulatory role in the innate host immune response through selective species-specific cytokine up-regulation and multiple signaling pathways.

Host response and immunology

The actual understanding of immunity to Tv has come from observations of responses in human patients and experimentation using in vitro models and animal models of the related species, T. foetus. Natural infection seems to produce immunity that is only partially protective, since reinfection of patients can be 30% on follow-up ⁸⁶. As it was mentioned before, most of Tv infection is often asymptomatic and recurrent, despite the presence of specific antibodies, suggesting the importance of the innate immune defense. Tv adhesion proteins, cysteine proteases, and the major parasite LPG play distinct roles in the pathogenesis and evasion of host immunity. LPG plays a key role in the parasite adherence and signaling to human vaginal and cervical epithelial cells, which is at least in part mediated by galectins. The epithelial cells respond to Tv infection and purified LPG by selective up-regulation of pro-inflammatory mediators. At the same time, Tv triggers an immunosuppressive response in monocytes, macrophages, and dendritic cells ¹¹⁷.

Evidence and mechanism

Antibody responses during Tv infection have been shown to occur in infected patients by several types of immunoassays ¹²⁹. Infection in humans produces parasite-specific antibodies in the reproductive tract and, in most instances, circulating antibodies in the serum. There is also evidence of lymphocyte priming as detected by antigen-specific proliferation of peripheral blood mononuclear cells ¹³⁰. Natural infection with Tv results in priming of acquired immune responses. The precise protective effects of parasite-specific immunity has not been so simple. In vitro analyses of the effects of serum-derived antibodies and monoclonal antibodies to surface antigens have identified anti-adhesin antibodies that block the adhesion of Tv to various human cell lines, such as HeLa ¹³¹, and to VECs ⁵¹. However, proof of the protective nature of antibodies in eliminating infection or limiting pathogenesis in vivo has been difficult because of lack of an adequate experimental-animal model for vaginal infection studies.

Targets of acquired immunity

The presence of parasite-specific immunoglobulin G ¹³² and immunoglobulin A ¹³³ responses, also indicates priming of helper T cells, although the relevant antigens and the exact effects of antibodies on the parasites are unknown. One obvious target of protective antibody could be the adhesin molecules used by the parasite to facilitate close contact to host cells, a process previously shown to lead to efficient host cell destruction ¹³⁴. The molecular basis of adhesion of Tv has been investigated, and four antigenic surface molecules have also been implicated in the adhesion of Tv to VECs; their expression is being up-regulated during attachment to host cells ⁷⁵.

Antibodies to these molecules protected target cells from parasite-mediated cytotoxicity ⁷⁵, suggesting that anti-adhesion immune responses could be important in vivo protection against the pathogenic effects of Tv. However, actual understanding of immunity to Tv remains unclear. It is not known whether acquired immune responses are required for protection and, if it is true what role is played by acquired immunity in containing or eliminating infections ⁸⁸. There is not a strong protective immunity after a natural infection in humans ⁸⁸.

Clinical features

The majority of women (85%) ¹³⁵ and men (77%) ¹³⁶ with Tv are asymptomatic. One third of asymptomatic women become symptomatic within six months ²¹. Among symptomatic men, symptoms include urethral discharge and dysuria. Among symptomatic women, common sites of infection include the vagina, urethra and endocervix. Symptoms include vaginal discharge (which is often diffuse, malodorous, yellow-green), dysuria, vaginal and/or vulvar itching, vulvar irritation, vaginal odor and abdominal pain. The normal vaginal pH is 4.5, but with Tv infection increases this markedly, often to >5 ^21,31,32. Colpitis macularis or strawberry cervix (microscopic, punctate haemorrhages in the cervix) is seen in about 2-5 % of women, though with colposcopy this rises to nearly 50% ^137,138 and the cervix becomes erythematosus, edematous, fragile and bleeds easily ¹³⁹. Other complications include infection of the adnexa, endometrium, Skene and Bartholin glands. In men, it can cause epididymitis, prostatitis, and decreased sperm cell motility ¹⁴⁰. When symptoms arise, the most common complaint among women diagnosed with Tv is vaginal discharge, presented in more than 50% of cases, followed by pruritus or dysuria ¹³⁹. The vaginal discharge is a classical signal of trichomoniasis and it is due to intense leukocytic infiltration within the genital tract because of the death of epithelial cells, which promotes inflammation and leads to an increased number of polymorphonuclear leukocytes in the vaginal fluid ¹⁴⁰. It is important to highlight that the infection symptoms are cyclic and more intense around the menses period because of the effect of iron on parasite pathogenesis ²¹. Studies have shown an association between Tv and vaginitis, cervicitis, urethritis, bacterial vaginosis, candidiasis, herpes simplex virus type-1 and type-2, chlamydia, gonorrhea, syphilis, human papillomavirus (HPV) and human immunodeficiency virus ^32,37. In addition, endometritis, adnexitis, pyosalpinx, and atypical pelvic inflammatory disease are related to Tv infection ¹¹⁷. Trichomoniasis may also affect the pregnancy course, causing low birth weight, premature rupture of membranes and preterm delivery ¹⁴¹. There are some evidence that Tv infection can be transmitted vertically leading to cases of vaginal and respiratory infections in neonates ^142,143. Mann et al. ¹⁴⁴ showed an association between maternal Tv infection and intellectual disability in children.

Inflammation and tumorigenesis

Over the past two decades, our understanding of inflammation in tumorigenesis has led to the identification of a number of molecules that are strongly linked to the development of human cancers ^145-147. Like tumorigenesis, tumor-promoting inflammation and tumor-associated inflammation (TAI) are the phenotypic product of a complex set of cellular and molecular interactions that result in an imbalance in the local microenvironment that is most analogous to an unresolved 'wound-healing' response ¹⁴⁷.

A number of the cellular and molecular mechanisms involved in inflammation-induced tumor initiation, promotion and progression are now well described (Table III). In recent years, strong evidence from many epidemiological and experimental studies have demonstrated that inflammation also plays an important role in the development of CIN and cervical cancer ¹⁴⁸. Cervical inflammation may be associated and may be a cofactor in high-grade cervical lesions in women infected with oncogenic HPV. In other cancer types, independent of the causal agent, chronic inflammation exposes the tissue to constant genotoxic damages. In cervical cancer, chronic inflammation added to the initial changes caused by high-risk HPV, may contribute to viral persistence and disease progression ^149,150.

Acute versus chronic inflammation and carcinogenesis

Acute inflammation has two balanced and biologically opposing effector arms or mechanisms: 1. a pro-apoptotic or tumoricidal and 2. a wound healing or pro-tumorigenic relationship model, where immune cells participate with the non-immune cells in the local environment (e.g. epithelial, vasculature and neuronal) ^151,152. Local or systemic adaptive immune responses (cell-mediated and humoral immunity) are activated and mobilized by selective signaling between the activated innate immune effector cells (e.g. macrophages and mast cells) and their counterparts in the adaptive immune system (e.g. T and B lymphocytes). In the acute inflammation, immune cells possess properties specialized that allowing them to recognize and eliminate intrinsic or extrinsic foreign elements and that injure or damage host tissue (acute phase). In the intermediate or resolution phase response, the immune cells function to resolve inflammation and repair the damaged tissue. Unresolved and persistent inflammation has been described as the loss of, or deregulation in the balance between both mechanisms. The role of persistent inflammation as a contributing factor in tumorigenesis is well accepted and, in many cancers, thought to be a necessary component. Examples include a causal relationship between inflammation and infectious agent-associated cancers [e.g. hepatitis B and C virus (liver), human papilloma virus (e.g. cervix, anal) and the bacterium Helicobacter pylori (stomach)]. The relationship between cancer and inflammation is also supported by the elevated risk of cancer in chronic inflammatory conditions, such as colitis-associated colorectal cancer. Importantly, the cause-effect relationship between inflammation and cancer is a challenging concept as it implies that inflammation precedes the processes. However, current evidence widely suggests that in the case of cancer, which is a multi-step and complex process, inflammation is an integral component of the overall pathogenesis of disease at the microenvironment level that not only contributes in a causal way but also supports a permissive state for tumors to grow ¹⁴⁵. It is important to recognize that TAI in solid tumors is itself a complex pathologic process, with contributions from classic immune cells as well as poorly characterized, cancer-associated fibroblasts and the epithelial tumor cell compartment ¹⁵¹. As it was mentioned before, Tv is infected by four different types of dsRNA viruses. When the parasite dies, releases its viral load within the human vagina. This could explain why symptoms can worsen upon successful antibiotic treatment and why Tv infected by the virus contribute to increase the inflammatory response and the virulence ¹⁵³.

Trichomonas vaginalis and cervical cancer

Epidemiology of cervical cancer

In 2018, according to the International Agency for Research on Cancer/Globocan (IARC), the cervical cancer (CC) was the ninth more frequent type of cancer worldwide, including both sexes, with 569,847 new cases, representing an incidence of 3.2% among all the cancers, and caused 311,365 deaths (incidence:3.3%), being the ninth deadliest cancer worldwide (IRC). CC represents the second most common female cancer, after breast cancer, worldwide ¹⁵⁴. The Pan-American Heath Organization/ World Health Organization (PAHO/WHO) ¹⁵⁵ reported 72,000 new cases of CC and almost 34,500 deaths in the American continent during 2018. The mortality rate was three times higher in Latin-America and the Caribbean area than in North-America ¹⁵⁵.

It is well known that the development of pre-malignant and malignant cervical lesions is multi-factorial. One of this most important factor is HPV, especially the high risk-HPV (hr-HPV) type 16 and 18, as the main etiological agent. Other risk factors have been identified in various cross-sectional and prospective cohort studies, including number of sexual partners (lifetime and recent), early onset of first intercourse, promiscuos partner, smoking, long-term use of oral contraceptives, multiparity, vaginal infection, malnutrition, certain dietary deficiencies, immunosuppressive conditions such HIV, and chronic inflammation due to co-infection with others microorganisms Ct, HSV-2, Ca, gonorrhea, BV, Ureaplasma urealyticum, Ureaplasma parvum, and Tv^156-158. Different authors ^159,160 have mentioned that the association among these STIs and cervical lesions may be related to their inflammation effects. Infection with hr- HPV plays a central role in cervical carcinogenesis. Epidemiological and molecular investigations have shown unequivocally that hr-HPV infection is the main causal factor in initiating the progressive transformation that leads to cervical intraepithelial neoplasia and to cervical cancer ^161-163. Many reports have demonstrated that HPV infections are mostly transitory and that only a small percentage of infections persist and may progress to dysplastic lesions: type-specific HPV persistence is the strongest risk factor for cervical cancer ¹⁶⁴. The reasons for the variable natural history remain poorly understood, but it has been generally accepted that different cofactors are involved in the development of dysplasia in HPV infected women. Factors, such as specific HPV genotype, viral load, and co-infection with more HPV types, are likely to be involved in the progression to precancerous cervical lesions ^164-167. In addition, environmental co-factors mentioned before have also been investigated as potentially involved in the disease process ^168-170. The sexually transmitted pathogens were reported to interact with HPV as cofactors in the progression of cervical neoplasias. For instance, Ct and Tv were proved to be positively correlated with hr-HPV persistent infection ¹⁷¹.

Trichomonas vaginalis/HPV/cervical intraephitelial neoplasia

Another important issue regarding complications of trichomoniasis in women is its involvement with an increased risk of cervical cancer. Some studies have pointed Tv as a predictor for cervical neoplasia since there is a high relative risk of preinvasive lesion and invasive cancer (CIN) ^8,172. As it was mentioned before, the cervical epithelium is a protective barrier which is disrupted by inflammation produced by Tv infection, allowing the penetration of hr-HPV to the basement membrane ^{8,159,163,172,173}. As it was mentioned in this manuscript before, Goshi et al.¹⁷² reported that the association of Tv with high grade CIN is because of the epithelial alteration and damage that characterize these conditions facilitate the proliferation of the organism. The cell mediated immunity generated against the invading organism involves recruitment of leucocytes in large numbers, which are associated with findings on cytology of smears. Further, the parasite often imbibes nutritious elements like fatty acids and iron by destruction of host red blood cells, which is caused by cytotoxic trypsin like substances called cell detaching factor, CDF and N-nitrosamines liberated during infection, which also promote the process of epithelial atypia and dysplasia. Vaginal pH rises during Tv infection, which allows the growth of the organism. Tv thrives on tissue debris and serous exudate and produces tissue damage that is extensive and atypical. Disease pathogenesis of Tv infection necessitates multitude of cross-communications involving viruses, bacteria, eukaryotes and human host ¹⁷⁴. Gram et al.¹⁷⁵ mentioned that the infections by HPV and Tv play an important role in the pathogenesis or CIN and CC. Lazenby et al. ⁸ had found Tv using nucleic acid amplification test (NAAT) to be associated with an increased risk of acquisition of high risk HPV (OR 4.2, 95% C.I. 1.7-10.3). Donders et al. ¹⁷⁶ demonstrated that HSIL cases where Tv was discovered, hr-HPV was present. Theses authors ¹⁷⁶ found that women with ASC-US with Tv had no HPV (36%). Although, the presence of Tv was higher in patients HPV negative/ASC-US compared to the group HPV positive (low and high risk)/ ASC-US patients. Tao et al.¹⁷⁷ found that TV infection is associated with CIN especially to high degree. They ¹⁷⁷ mentioned that there is some epidemiological evidence to suggest that genital tract disease such as cervical inflammation might be linked to cervical cancer or high-grade lesions, and a significant association between cervical inflammation and HISL was identified in this study. Tv may act as a cofactor facilitating the development of cervical HPV infection to high-grade lesion and cervical cancer. Multiple studies have demonstrated an association between previous and current Tv infection and cervical dysplasia and human papillomavirus ^172,178. Fem et al.¹⁷⁹ found that women with HPV are more likely to be infected by other types of STIs. Also, they said Tv+ women had an increased risk to get hr-HPV infection compared to negative Tv women. Yap et al.¹⁸⁰ found antibodies against Tv in 41.3% of patients with invasive CC compared with 5% in control group.

CONCLUSION

Tv is considered as the second most frequent STIs and the second most common cause of lower genital tract infection worldwide. Because Tv infection is highly prevalent in sexually active populations, it is now gaining greater recognition as an important source of reproductive morbidity and is clearly associated with significant public health problems. Also, Tv infection has the potential to increase the possibility for the acquisition and transmission of HIV, possibly HSV-2 and HPV. It has been independently associated with cancer, so it has been considered as a co-factor or factor for CC. Trichomoniasis is clearly associated with significant public health problems. The disease presentation is variable and diagnosis can be difficult with the most commonly used methods. Certain socio-demographic and behavioral risk factors may assist in predicting infection. A majority of women and a significant proportion of men with trichomoniasis are asymptomatic; these patients would thus escape detection and treatment under syndromic management recommendations. Control programs must work towards increased screening in order to have any effect on the burden of this disease. In recent years, many advances have been made in the epidemiology, diagnosis, and treatment of Tv. The focus of these efforts, however, has largely been on women. Although there are still many open questions regarding Tv's epidemiology, particularly in the context of facilitated HIV contagion and cancer, our understanding of Tv's pathogenesis made a large leap forward and the picture is becoming ever more complete. Finally, the Tv genome has remained as a fascinating field of study. So far, there have been four dsRNA viruses identified. This area opens a great and broad field for research about the virulence and its possible role as co-factor or factor in CC in the next years to come.