Trichomonas vaginalis extracellular vesicles up-regulate and directly transfer adherence factors promoting host cell colonization.

Trichomonas vaginalis, a common sexually transmitted parasite that colonizes the human urogenital tract, secretes extracellular vesicles (TvEVs) that are taken up by human cells and are speculated to be taken up by parasites as well. While the crosstalk between TvEVs and human cells has led to insight into host:parasite interactions, roles for TvEVs in infection have largely been one-sided, with little known about the effect of TvEV uptake by T. vaginalis. Approximately 11% of infections are found to be coinfections of multiple T. vaginalis strains. Clinical isolates often differ in their adherence to and cytolysis of host cells, underscoring the importance of understanding the effects of TvEV uptake within the parasite population. To address this question, our lab tested the ability of a less adherent strain of T. vaginalis, G3, to take up fluorescently labeled TvEVs derived from both itself (G3-EVs) and TvEVs from a more adherent strain of the parasite (B7RC2-EVs). Here, we showed that TvEVs generated from the more adherent strain are internalized more efficiently compared to the less adherent strain. Additionally, preincubation of G3 parasites with B7RC2-EVs increases parasite aggregation and adherence to host cells. Transcriptomics revealed that TvEVs up-regulate expression of predicted parasite membrane proteins and identified an adherence factor, heteropolysaccharide binding protein (HPB2). Finally, using comparative proteomics and superresolution microscopy, we demonstrated direct transfer of an adherence factor, cadherin-like protein, from TvEVs to the recipient parasite's surface. This work identifies TvEVs as a mediator of parasite:parasite communication that may impact pathogenesis during mixed infections.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis.

BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

A riboside hydrolase that salvages both nucleobases and nicotinamide in the auxotrophic parasite Trichomonas vaginalis.

Pathogenic parasites of the Trichomonas genus are causative agents of sexually transmitted diseases affecting millions of individuals worldwide and whose outcome may include stillbirths and enhanced cancer risks and susceptibility to HIV infection. Trichomonas vaginalis relies on imported purine and pyrimidine nucleosides and nucleobases for survival, since it lacks the enzymatic activities necessary for de novo biosynthesis. Here we show that T. vaginalis additionally lacks homologues of the bacterial or mammalian enzymes required for the synthesis of the nicotinamide ring, a crucial component in the redox cofactors NAD+ and NADP. Moreover, we show that a yet fully uncharacterized T. vaginalis protein homologous to bacterial and protozoan nucleoside hydrolases is active as a pyrimidine nucleosidase but shows the highest specificity toward the NAD+ metabolite nicotinamide riboside. Crystal structures of the trichomonal riboside hydrolase in different states reveals novel intermediates along the nucleoside hydrolase-catalyzed hydrolytic reaction, including an unexpected asymmetry in the homotetrameric assembly. The active site structure explains the broad specificity toward different ribosides and offers precise insights for the engineering of specific inhibitors that may simultaneously target different essential pathways in the parasite.

Lactobacillus gasseri and Gardnerella vaginalis produce extracellular vesicles that contribute to the function of the vaginal microbiome and modulate host-Trichomonas vaginalis interactions.

Trichomonas vaginalis is an extracellular protozoan parasite of the human urogenital tract, responsible for a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a dysbiotic microbiome that is characterised by the depletion of host-protective commensals such as Lactobacillus gasseri, and the flourishing of a bacterial consortium that is comparable to the one seen for bacterial vaginosis, including the founder species Gardnerella vaginalis. These two vaginal bacteria are known to have opposite effects on T. vaginalis pathogenicity. Studies on extracellular vesicles (EVs) have been focused on the direction of a microbial producer (commensal or pathogen) to a host recipient, and largely in the context of the gut microbiome. Here, taking advantage of the simplicity of the human cervicovaginal microbiome, we determined the molecular cargo of EVs produced by L. gasseri and G. vaginalis and examined how these vesicles modulate the interaction of T. vaginalis and host cells. We show that these EVs carry a specific cargo of proteins, which functions can be attributed to the opposite roles that these bacteria play in the vaginal biome. Furthermore, these bacterial EVs are delivered to host and protozoan cells, modulating host-pathogen interactions in a way that mimics the opposite effects that these bacteria have on T. vaginalis pathogenicity. This is the first study to describe side-by-side the protein composition of EVs produced by two bacteria belonging to the opposite spectrum of a microbiome and to demonstrate that these vesicles modulate the pathogenicity of a protozoan parasite. Such as in trichomoniasis, infections and dysbiosis co-occur frequently resulting in significant co-morbidities. Therefore, studies like this provide the knowledge for the development of antimicrobial therapies that aim to clear the infection while restoring a healthy microbiome.

Vaginitis and risk of sexually transmitted infections: results of a multi-center U.S. clinical study using STI nucleic acid amplification testing.

Significant increases in rates of sexually transmitted infections (STIs) caused by Trichomonas vaginalis (TV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) are occurring in the United States. We present results of a U.S. study examining the intersection of STIs and vaginitis. Among 1,051 women with diagnoses for the presence or absence of bacterial vaginosis (BV) and/or symptomatic vulvovaginal candidiasis (VVC), 195 (18.5%) had one or more STIs, including 101 (9.6%) with TV, 24 (2.3%) with CT, 9 (0.8%) with NG, and 93 (8.8%) with MG. STI prevalence in BV-positive women was 26.3% (136/518), significantly higher than STI prevalence of 12.5% (59/474) in BV-negative women (P < 0.0002). Unlike infections with CT or NG, solo infections of MG or TV were each significantly associated with a diagnosis of BV-positive/VVC-negative (OR 3.0751; 95% CI 1.5797-5.9858, P = 0.0113, and OR 2.873; 95% CI 1.5687-5.2619, P = 0.0017, respectively) and with mixed infections containing MG and TV (OR 3.4886; 95% CI 1.8901-6.439, P = 0.0042, and OR 3.1858; 95% CI 1.809-5.6103, P = 0.0014, respectively). TV and MG infection rates were higher in all Nugent score (NS) categories than CT and NG infection rates; however, both STIs had similar comparative prevalence ratios to CT in NS 6-10 vs NS 0-5 (CT: 3.06% vs 1.4%, 2.2-fold; MG: 10.7% vs 6.1%, 1.8-fold; TV: 14.5% vs 7.0%, 2.1-fold). NG prevalence was relatively invariant by the NS category. These results highlight the complexity of associations of STIs with two major causes of vaginitis and underscore the importance of STI testing in women seeking care for abnormal vaginal discharge and inflammation. IMPORTANCE: This study reports high rates for sexually transmitted infections (STIs) in women seeking care for symptoms of vaginitis and bacterial vaginosis, revealing highly complex associations of STIs with two of the major causes of vaginal dysbiosis. These results underscore the importance of STI testing in women seeking care for abnormal vaginal discharge and inflammation.

Sandwich enzyme-linked aptamer-based assay for the detection of Trichomonas vaginalis.

Trichomoniasis is the most prevalent curable, non-viral sexually transmitted infection (STI), with an estimated 156 million new infections in 2020. It can potentially result in adverse birth outcomes as well as infertility in men, whilst it also increases the risk of acquiring HIV and contracting other vaginal infections. It is mostly prevalent among women in low-income countries and especially in Africa and the Americas. This STI is caused by Trichomonas vaginalis (TV) and a robust, cost-effective, sensitive, specific and rapid diagnostic test is urgently required. We report the screening of 6 full-length and 4 truncated aptamers previously selected in our group for use in a microplate-based sandwich assay. The combination of dual aptamers comprising a short 14-mer truncated capture aptamer (termed A1_14mer) and a full-length non-truncated reporter aptamer (A6) was elucidated to be the optimum pair for a sensitive sandwich enzyme-linked aptamer assay (ELAA) for the detection of TV achieving a detection limit of 3.02 × 104 TV cells/mL. The results obtained with the A1_14mer-A6 ELAA correlate excellently with wet-mount microscopy for the detection of TV in clinical specimens, cervicovaginal lavages and vaginal swabs, highlighting the potential clinical application of this assay for cost-effective population screening and subsequent prevention of the onset of complications associated with undiagnosed and untreated TV.

Complement receptor 3 is required for maximum in vitro trogocytic killing of the parasite Trichomonas vaginalis by human neutrophil-like cells.

Trichomonas vaginalis (Tv) is a parasite that causes trichomoniasis, a prevalent sexually-transmitted infection. Neutrophils are found at the site of infection, and can rapidly kill the parasite in vitro, using trogocytosis. However, the specific molecular players in neutrophil killing of Tv are unknown. Here, we show that complement proteins play a role in Tv killing by human neutrophil-like cells (NLCs). Using CRISPR/Cas9, we generated NLCs deficient in each of three complement receptors (CRs) known to be expressed on human neutrophils: CR1, CR3, and CR4. Using in vitro trogocytosis assays, we found that CR3, but not CR1 or CR4 is required for maximum trogocytosis of the parasite by NLCs, with NLCs lacking CR3 demonstrating ~40% reduction in trogocytosis, on average. We also observed a reduction in NLC killing of Tv in CR3 knockout, but not CR1 or CR4 knockout NLCs. On average, NLCs lacking CR3 had ~50% reduction in killing activity. We also used a parallel approach of pre-incubating NLCs with blocking antibodies against CR3, which similarly reduced NLC killing of parasites. These data support a model in which Tv is opsonized by the complement protein iC3b, and bound by neutrophil CR3 receptor, to facilitate trogocytic killing of the parasite.

Detection of Trichomonas vaginalis by Allplex™ STI Essential Assay (Seegene) in clinical samples from the Brazilian public health system users.

The failures in Trichomonas vaginalis (TV) infection diagnosis leave more than half of cases unidentified. In this report, urine and vaginal discharge samples were analyzed by wet mount, culture examination, and real-time PCR by Allplex™ (Seegene®) kit, in a population assisted by the Brazilian Public Health System. From 747 samples, 2.81% were positive for TV in wet mount and culture, and 3.88% by Allplex™. Samples kept at - 80 ºC for 22 months did not impair the PCR technique. The sensitivity for wet mount, culture, and Allplex™ was 72, 100, and 100%, respectively. Allplex™ technique showed highest detection of TV.

Trichomonas vaginalis: comparison of primers for implementation as an in-house PCR in rural Vellore, South India.

BACKGROUND: Trichomonas vaginalis (TV) accounts for the highest burden of curable, non-viral sexually transmitted infections worldwide. Prevalence in India ranges from 0.4 to 27.4% in women and 0.0-5.6% in men. In 2015, the prevalence of TV among pregnant women of rural Vellore was 3.11% using Sekisui OSOM® Trichomonas test and culture methods. Molecular methods are the most sensitive, rapid diagnostic tool for Sexually Transmitted Infection's (STI) albeit cost hinders implementation of commercial platforms. To determine a sensitive, sustainable molecular method, we compared three targets (Adhesin AP65, cytoskeleton Beta-tubulin BTUB 9/2 and TVK 3/7) with the highest published diagnostic accuracy against microscopy, culture and Real Time PCR (RT- PCR). MATERIALS & METHODS: Six-hundred adult, sexually active women attending the Obstetrics-Gynaecology rural out-patient clinic the Rural Unit for Health and Social Affairs (RUHSA) from July 2020 - February 2021 were enrolled. A vaginal lateral and posterior fornix specimen was inoculated, onsite, into Biomed InPouch® TV culture and smeared onto a slide for fluorescence microscopy using Acridine orange. A flocked nylon swab specimen for PCR was used to determine the sensitivities of the Adhesin AP65, cytoskeleton Beta-tubulin BTUB 9/2 and TVK 3/7 gene targets. Seegene Allplex™ STI Essential Assay, S.Korea was used to confirm TV positives. RESULTS: Nine specimens (9/600, 1.5%) were positive for TV. There was a 100% correlation between Biomed InPouch TV® culture, PCR with TVK 3/7 and RT-PCR while a correlation of 66.6% with BTUB 9/2 and AP65 gene targets. Clinically, 77.7% (n = 7) presented with white-greenish discharge per vagina, 11% (n = 1) with infertility, 22.2% (n = 2) were asymptomatic. Eight of nine patients (88.9%) had co-infections with other bacterial STIs. Prevalence of TV coinfection with Neisseria gonorrhoea was 1.1%. CONCLUSION: Current hospital-based prevalence of TV in rural Vellore was 1.5%. Repetitive DNA target TVK 3/7 was more sensitive than AP65 and BTUB 9/2 primers.

Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases.

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted ß-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.

Bacterial vaginosis (BV) and Trichomonas vaginalis (TV) co-infection, and bacterial antibiogram profile of pregnant women studied in Lagos, Nigeria.

AIM: This study was undertaken to determine the prevalence of Bacterial Vaginosis (BV), Trichomonas Vaginalis (TV) co-infection, and the antibacterial sensitivity profile of bacterial isolates. METHODS: The study was a cross-sectional study of 232 pregnant women on a routine antenatal visit between April 2019 and Sept. 2020, at Amukoko clinic in Lagos, Nigeria. The gynaecologist conducted the clinical examination on each patient looking for vaginal discharge and its consistency/homogeneity, colour and odour. Two High Vaginal Swab (HVS) samples were taken from every patient and a semi-structured questionnaire was used to gather the socio-demographic, practices/attitudes, and clinical information of each participant. One sample was employed for wet preparation to identify the TV and BV diagnosis using Amsel's criteria and Whiff's test. The second sample was used for bacterial culture and antibiogram was conducted using the disc diffusion technique. The Clinical Laboratory Standard Institutes' (CLSI) interpretative criteria were used to categorise the results. RESULTS: The mean age of the clients was 28.11 ± 7.08 years of age. The majority (88%) were aged 15-35 years. Only 81 (34.9%) had microbial organisms isolated or seen from their specimens and 19 (8.2%) of such were classified as having BV (Bacteriods or Gardnerella isolated). Of the 81 infected, 33 (40.8%) had only bacterial infection, 36 (44.4%) had TV alone and 12 (14.8%) had bacteria co-infected with TV. From the clinical records, the population that was classified as having UTI or vaginitis was only 46 (20.7%) The study observed age (15-35 years) related association between vaginosis/ TV co-infection (X2 = 7.9; P = 0.005). Participants with symptoms of vaginitis or UTI (mainly E. coli & pseudomonas spp. isolated), BV/co-infection with TV significantly associated with female traders (X2 = 8.5; P = 0.003) and were more associated with those from polygamous relationships (X2 = 18.79, P = 0.0001). Women in their 3rd and 2nd. trimester were more significantly associated with vaginal infection (X2 = 9.47, P = 0.002; X2 = 4.79, P = 0.029) respectively. The Pseudomonas showed susceptibility to ciprofloxacin (CIP) and cefuroxime (CXM). While, E. coli isolates were susceptible to cefepime, ciprofloxacin, and imipenem. CONCLUSION: There is a relatively low prevalence of BV and flagellate co-infection in the community studied. RECOMMENDATION: We recommend screening of antenatal women with underlying symptoms for BV and flagellates co-infection to avoid its progression to vaginitis.

The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes.

BACKGROUND: The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. RESULTS: Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. CONCLUSIONS: Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

The presence of Trichomonas vaginalis in urogenital samples can affect the sensitivity of Mycoplasma hominis identification techniques, leading to an underestimation of bacterial infections.

Trichomonas vaginalis and Mycoplasma hominis, two microorganisms causing infections of the urogenital tract, are closely associated in that they establish an endosymbiosis relationship, the only case among human pathogens. As a result, the presence of one microorganism may be considered a sign that the other is present as well. Identification of the two pathogens in clinical samples is based on cultivation techniques on specific media, even though in recent years, new sensitive and rapid molecular techniques have become. Here, we demonstrate that the concomitant presence of T.vaginalis in urogenital swabs may lead to a delay in the identification of M.hominis, and thus to an underestimation of bacterial infections when cultural techniques are used.

Transcriptional profile of Trichomonas vaginalis in response to metronidazole.

BACKGROUND: Trichomoniasis caused by Trichomonas vaginalis, combined with its complications, has long frequently damaged millions of human health. Metronidazole (MTZ) is the first choice for therapy. Therefore, a better understanding of its trichomonacidal process to ultimately reveal the global mechanism of action is indispensable. To take a step toward this goal, electron microscopy and RNA sequencing were performed to fully reveal the early changes in T. vaginalis at the cellular and transcriptome levels after treatment with MTZ in vitro. RESULTS: The results showed that the morphology and subcellular structures of T. vaginalis underwent prominent alterations, characterized by a rough surface with bubbly protrusions, broken holes and deformed nuclei with decreased nuclear membranes, chromatin and organelles. The RNA-seq data revealed a total of 10,937 differentially expressed genes (DEGs), consisting of 4,978 upregulated and 5,959 downregulated genes. Most DEGs for the known MTZ activators, such as pyruvate:ferredoxin oxidoreductase (PFOR) and iron-sulfur binding domain, were significantly downregulated. However, genes for other possible alternative MTZ activators such as thioredoxin reductase, nitroreductase family proteins and flavodoxin-like fold family proteins, were dramatically stimulated. GO and KEGG analyses revealed that genes for basic vital activities, proteostasis, replication and repair were stimulated under MTZ stress, but those for DNA synthesis, more complicated life activities such as the cell cycle, motility, signaling and even virulence were significantly inhibited in T. vaginalis. Meanwhile, increased single nucleotide polymorphism (SNP) and insertions - deletions (indels) were stimulated by MTZ. CONCLUSIONS: The current study reveals evident nuclear and cytomembrane damage and multiple variations in T. vaginalis at the transcriptional level. These data will offer a meaningful foundation for a deeper understanding of the MTZ trichomonacidal process and the transcriptional response of T. vaginalis to MTZ-induced stress or even cell death.

Clinical Evaluation of a New Molecular Test for the Detection of Organisms Causing Vaginitis and Vaginosis.

In this prospective, observational, method comparison clinical study, the Xpert Xpress MVP test (MVP) was evaluated using both clinician-collected (CVS) and self-collected vaginal swabs (SVS) collected in a clinical setting. The study was conducted at 12 sites, including point-of-care (POC) settings, from geographically diverse locations in the United States. Participants were biologically female patients ≥ 14 years old with signs and/or symptoms of vaginitis/vaginosis. MVP test results for BV were compared to the BD MAX Vaginal Panel (BDVP). Results for Candida group and Candida glabrata and Candida krusei targets (species not differentiated) were assessed relative to yeast culture followed by mass spectrometry for species identification. Trichomonas vaginalis (TV) results were compared relative to a composite method that included results from the BDVP and InPouch TV culture. The investigational test demonstrated high positive percent agreement ranging from 93.6 to 99.0%, and negative percent agreement ranging from 92.1% to 99.8% for both CVS and SVS specimens, indicating it may be a valuable tool for the diagnosis of vaginitis/vaginosis in laboratory and POC settings.

Disparities in adherence to retesting guidelines in women with Trichomonas vaginalis infection.

BACKGROUND: Trichomoniasis is the most prevalent nonviral sexually transmitted infection in the United States. Numerous studies have shown disproportionately higher prevalence rates in non-Hispanic Black women. Because of the high rates of reinfection, the Centers for Disease Control and Prevention recommends retesting women treated for trichomoniasis. Despite these national guidelines, there are few studies examining adherence to retesting recommendations for patients with trichomoniasis. Adherence to retesting guidelines has been shown in other infections to be an important determinant of racial disparities. OBJECTIVE: This study aimed to describe Trichomonas vaginalis infection rates, evaluate adherence to retesting guidelines, and examine characteristics of women who were not retested according to the guidelines in an urban, diverse, hospital-based obstetrics and gynecology clinic population. STUDY DESIGN: We conducted a retrospective cohort study of patients from a single hospital-based obstetrics and gynecology clinic who were tested for Trichomonas vaginalis between January 1, 2015 and December 31, 2019. Descriptive statistics were used to examine guideline-concordant testing for reinfection among patients with trichomoniasis. Multivariable logistic regression was used to identify characteristics associated with testing positive and with appropriate retesting. Subgroup analyses were performed for patients who were pregnant and tested positive for Trichomonas vaginalis. RESULTS: Among the 8809 patients tested for Trichomonas vaginalis, 799 (9.1%) tested positive at least once during the study. Factors associated with trichomoniasis included identifying as non-Hispanic Black (adjusted odds ratio, 3.13; 95% confidence interval, 2.52-3.89), current or former tobacco smoking (adjusted odds ratio, 2.27; 95% confidence interval, 1.94-2.65), and single marital status (adjusted odds ratio, 1.96; 95% confidence interval, 1.51-2.56). Similar associated factors were found in the pregnant subgroup analysis. For women with trichomoniasis, guideline-concordant retesting rates were low across the entire population, with only 27% (214/799) of patients retested within the recommended time frame; 42% (82/194) of the pregnant subgroup underwent guideline-concordant retesting. Non-Hispanic Black women had significantly lower odds of undergoing guideline-recommended retesting than non-Hispanic White women (adjusted odds ratio, 0.54; 95% confidence interval, 0.31-0.92). Among patients tested according to guideline recommendations, we found a high rate of Trichomonas vaginalis positivity at retesting: 24% in the entire cohort (51/214) and 33% in the pregnant subgroup (27/82). CONCLUSION: Trichomonas vaginalis infection was identified at a high frequency in a diverse, urban hospital-based obstetrics and gynecology clinic population. Opportunities exist to improve on equitable and guideline-concordant retesting of patients with trichomoniasis.

Inflammatory responses during trichomoniasis: The role of Toll-like receptors and inflammasomes.

Toll-like receptors (TLRs) and inflammasomes belong to the pattern recognition receptors (PRRs) of innate immunity identifying conserved compounds produced by pathogens or discharged by injured cells. Different cell subsets in the human urogenital system, such as epithelial cells and infiltrating leukocytes, express different kinds of TLRs (such as TLR2, TLR3, TLR4, TLR5 and TLR9) as well as inflammasomes (such as NLRP3, NLRC4 and AIM2). Various types of the Trichomonas vaginalis-derived components such as glycosyl-phosphatidylinositol (GPI), T. vaginalis virus (TVV), Lipophosphoglycan (LPG) and flagellin can be recognized by TLR2, TLR3, TLR4 and TLR5, respectively, leading to the production of proinflammatory cytokines and chemokines in the cervicovaginal mucosa. The T. vaginalis-induced inflammasomes can lead to pyroptosis as well as the release of IL-1ß and IL-18 promoting innate and adaptive immune responses. The PRR-mediated responses to T. vaginalis may contribute to the induction of protective immune responses, local inflammation, promotion of co-infections, or even the development of malignancies, for example, prostate cancer. The protective or pathogenic roles of the TLRs and inflammasomes during trichomoniasis are highlighted in this review. A better understanding of PRR-mediated responses provides invaluable insights to develop effective immunotherapeutic strategies against T. vaginalis infection.

Construction a novel detection method for Trichomonas vaginalis based on recombinant enzyme polymerase amplification targeting the Actin gene.

Trichomoniasis is a common and curable sexually transmitted disease worldwide. The rapid, convenient, and accurate diagnosis of trichomoniasis is an important link in the prevention and treatment of the disease. The current detection methods of Trichomonas vaginalis are mainly wet mount microscopy, culture, nested PCR, and loop-mediated isothermal amplification. However, these detection methods have some shortcomings. In this study, a recombinant enzyme polymerase amplification (RPA) assay had been conducted to detect T. vaginalis. The target gene and the corresponding primers were screened, and the reaction system and conditions were optimized in the assay of RPA. The sensitivity and specificity of this detection method were analyzed. The detection efficiency of wet mount microscopy, culture, nested PCR, and RPA was compared by testing 53 clinical samples from vaginal secretions. By screening, the actin gene of T. vaginalis could be used as a target gene for RPA detection of T. vaginalis, and the optimum reaction condition to amplify the actin gene by RPA was at 39°C for 30 min. The detection limit of T. vaginalis DNA using RPA was 1 pg, corresponding to a sensitivity of approximately five trophozoites. The RPA assay demonstrated high specificity for T. vaginalis, and there was no cross-reactivity with Giardia lamblia, Escherichia coli, Lactobacillus, Toxoplasma gondii, Staphylococcus aureus, and Candida albicans. Of the 53 clinical samples, the positive rates of T. vaginalis detected by wet mount microscopy, culture, nested PCR and RPA were 50.9 4% (27/53), 71.7% (38/53), 71.7% (38/53), and 69.81% (37/53), respectively. Compared with culture which was used as the gold standard for diagnosing trichomoniasis, testing clinical samples by wet mount microscopy showed 71.05% sensitivity, 100% specificity, and moderate diagnostic agreement with the culture (K = 0.581, Z = 4.661, p < 0.001). The nested PCR showed 100% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 1, Z = 7.28, p < 0.001), while RPA displayed 97.37% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 0.954, Z = 6.956, p < 0.001). At the present study, rapid amplification of actin gene by RPA could be used as a tool for detection of T. vaginalis. The detection method of RPA was more sensitive than wet mount microscopy and displayed excellent specificity. Moreover, RPA amplification of actin gene did not require a PCR instrument and the amplification time was shorter than that of ordinary PCR. Therefore, the RPA assay was proposed in this study as a point-of-care examination and a diagnostic method of T. vaginalis infection, which exhibited the potential value in the treatment and prevention of trichomoniasis.

The prevalence of Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) at testing centers in Belgium, Germany, Spain, and the UK using the cobas TV/MG molecular assay.

Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) can lead to long-term sequelae in males and females; however, global prevalence data vary between geographical regions, as these sexually transmitted infections are not included in routine screening. The objective of this study was to use the cobas® TV/MG assay to assess the point prevalence of TV and MG in specimens from men and women over a broad European geographical area. Urine, vaginal, endocervical, and rectal samples were collected from patients aged ≥ 18 years receiving Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (NG) screening as per local standard of care at sites in Belgium, Germany, Spain, and the UK (Wales). Remnant samples were assessed using the cobas TV/MG assay. Analysis of 2795 samples showed that MG prevalence varied slightly across female sample types (range: 1.7-5.8%; p = 0.0042). MG prevalence was higher in male rectal samples (12.5%) than in male urine samples (3.9%; p < 0.0001). TV prevalence was low in male (0.8%; 12/1535) and female (1.3%; 16/1260) samples across all sites. Co-infection of TV/MG with CT or NG was 10.0% (19/190) and 9.6% (7/73), respectively, in both male and female samples. MG and TV prevalence rates were comparable to the published literature in Europe. MG prevalence was highest in male rectal samples; as rectal testing is an off-label use of the cobas TV/MG assay, the clinical utility of this assay for rectal testing should be further investigated.

Vaginal Swab vs Urine for Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis: A Meta-Analysis.

PURPOSE: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the 2 most frequently reported notifiable sexually transmitted infections (STIs) in the United States, and Trichomonas vaginalis (TV), although not a notifiable disease, is the most common curable non-viral STI worldwide. Women bear a disproportionate burden of these infections and testing is necessary to identify infections. Although vaginal swabs are the recommended sample type, the specimen most often used among women is urine. The objective of this meta-analysis was to assess the diagnostic sensitivity of commercially available assays for vaginal swabs vs urine specimens from women. METHODS: A systematic search of multiple databases from 1995 through 2021 identified studies that (1) evaluated commercially available assays, (2) presented data for women, (3) included data obtained from the same assay on both a urine specimen and a vaginal swab from the same patient, (4) used a reference standard, and (5) were published in English. We calculated pooled estimates for sensitivity and the corresponding 95% CIs for each pathogen as well as odds ratios for any difference in performance. RESULTS: We identified 28 eligible articles with 30 comparisons for CT, 16 comparisons for NG, and 9 comparisons for TV. Pooled sensitivity estimates for vaginal swabs and urine, respectively, were 94.1% and 86.9% for CT, 96.5% and 90.7% for NG, and 98.0% and 95.1% for TV (all P values <.001). CONCLUSIONS: Evidence from this analysis supports the Centers for Disease Control and Prevention's recommendation that vaginal swabs are the optimal sample type for women being tested for chlamydia, gonorrhea, and/or trichomoniasis.