MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 98(2) March 2003
PAGES: 273-276 DOI: Short communication
Perspective of a New Diagnostic for Human Trichomonosis

Sheila B Lecke, Tiana TascaI, André A SoutoII, Geraldo A De Carli +

Laboratório de Parasitologia Clínica, Faculdade de Farmácia
IDepartamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil
IILaboratório de Síntese Orgânica, Faculdade de Química, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brasil 

Abstract

Several diagnostic techniques have been employed for the detection of Trichomonas vaginalis. Microtubules constitute the cytoskeleton in eukaryotic cells and are sensitive to antimitotic drugs, such as Taxol (paclitaxel). We used FLUTAX a fluorescent taxoid _ to analyze the microtubule distribution in living trophozoites of T. vaginalis in urine and in vaginal discharge. A high intensity of fluorescence was observed in living T. vaginalis, epithelial cells and leukocytes present in urine and vaginal discharge. Our preliminary results show the perspective of a new diagnostic technique for trichomonosis and will contribute to the understanding of the cytoskeleton of T. vaginalis.

Trichomonas vaginalis is a flagellated parasitic protist of the human urogenital tract. The parasite is a common cause of infection in the female tract, and its clinical presentation ranges from a totally asymptomatic infection to a severe vaginitis. In men, the infection is mostly asymptomatic, but in some cases it can lead to a mild urethritis which usually resolves spontaneously within two weeks. Recent publications indicate that this parasite's impact is not only limited to vaginitis but also a major factor in promoting transmission of HIV (Sorvillo & Kerndt 1998, Fleming & Wasserheit 1999), in causing low-weight and premature birth (Lehker & Alderete 2000), and in predisposing women to atypical pelvic inflammatory disease (Heine & McGregor 1993), cervical cancer (Gram et al. 1992, Kharsany et al. 1993, Zhang & Begg 1994), and infertility (Grodstein et al. 1993). Like others trichomonads, T. vaginalis does not present a cystic form, only the trophozoitic one. The organism is ellipsoidal and presents four anterior flagella unequal in size, an undulating membrane, an axostyle, a nucleus, a Golgi apparatus and hydrogenosomes, instead of mitochondria. Trichomonosis is the most common non-viral sexually-transmitted disease (STD) in the world (Gerbase et al. 1998). Donné described the protozoan for the first time in 1836, however diagnosis of trichomonosis still presents some difficulties.

The diagnosis of this infection, based only on clinical aspects, such as characteristics of vaginal discharge, may be erroneous (Madico et al.1998). Various laboratory techniques have been employed for the detection of T. vaginalis. Positive diagnosis of trichomonosis is generally established by wet-mount examination, but this method is only 30-80% sensitive when compared to the gold standard of culture (Lehker & Alderete 2000). Conversely, culture is laborious, sometimes inaccessible and often cost prohibitive for many clinical settings, and is not routinely used (Heine & McGregor 1993, Heine et al. 1997). Different stains or cytochemical reactions including acridine orange, Giemsa, Leishman, Diff-Quick, Fontana, Gram, periodic acid-Schiff, iron hematoxilin and Papanicolau (De Carli 2001) have also been used in smears. A cost effective and sensitive alternative, with a similar clinical efficacy as traditional culture, is the InPoucht TV culture system (Ohlemeyer et al. 1998). This is a disposable culture system for the maintenance, transport, and detection of T. vaginalis in clinical specimens (Borchardt & Smith 1991). Other diagnostic techniques such as enzyme-linked immunosorbent assay (ELISA) (Sharma et al. 1991), hybridization (DeMeo et al. 1996) and fluorescent antibody test (Krieger 1988) have been used to detect the parasite. These procedures contribute to increase the certainty of diagnosis, but do not substitute the culture exams (Honigberg & Burgess 1994, De Carli 2001). The advent of the polymerase chain reaction (PCR), however, has opened a new avenue for diagnosis of T. vaginalis (Shaio et al. 1997, Madico et al. 1998).

In this study, we present a perspective of a new diagnostic technique for trichomonosis, based on the analysis of microtubule distribution in living trophozoites of T. vaginalis in urine and vaginal discharge, using the fluorescent taxoid Flutax-2. Flutax-2, described by Souto et al.(1995), is an active fluorescent derivative of Taxol (paclitaxel). Like Taxol, FLUTAX-2 binds to the polymerized ab-tubulin dimer, allowing direct and rapid observation by fluorescence microscopy (Diaz et al. 2000).

T. vaginalis presents a poorly known cytoskeleton formed by an axostyle, which is a trunk that courses from one end of the cell to the other oriented by microtubules (Benchimol et al. 2000), and a pelta, which like the axostyle, is also formed by stable structures such as microtubules (Ribeiro et al. 2000). Microtubules are essential for the maintenance of cell shape and its organization; for cytoplasm transport, motility and division in all eukaryotic cells.

Urine and vaginal secretions were observed in an hemocytometer to determine number of trophozoites, epithelial cells, leukocytes, artifacts and the presence of some erythrocytes and yeast. We used three strains in this study: HSL-1, recently isolated from women with trichomonosis in a State medical unit, and two American Type Culture Collection (ATCC) strains, 30236 (sensitive to metronidazole) and 30238 (resistant to metronidazole). The ATCC strains were cultivated axenically in vitro at 37°C, in trypticase-yeast extract-maltose (TYM), Diamond medium (Diamond 1957) without agar, pH 6.0, supplemented with 10% heat inactivated bovine serum, penicillin (1000 IU/ml) and streptomycin sulfate (1 mg/ml). Both strains were counted in an hemocytometer to obtain 106 cells/ml.

The positive urine and vaginal discharge from the patient (HSL-1), and ATCC strains were incubated with FLUTAX-2 (1 µM, final concentration) at 37°C, for 40 min, and observed with an Axiolab MC 80 DX (Zeiss) fluorescence microscope.

The photomicrographs showed a high intensity of fluorescence in the axostyle, centrossome and flagella of living cells of T. vaginalis,strongly suggesting the microtubule composition of these structures (Figs 1-3). Epithelial cells showed an homogenous fluorescence throughout the cytoplasm and surrounding the nucleus (Figs 4, 5). Prominent plasma membranes in the epithelial cells were particularly noted, suggesting that they are superimposed. Leukocytes were observed as small spheres with intensive fluorescence. No erythrocytes or yeast were observed. Under fluorescence microscope T. vaginalis was easily differentiated from other structures, since the parasitic trophozoite is alive, presenting flagella mobility, while all other cells are dead. In addition, the size of each structure represents another feature for identification of each organism. A difference in fluorescence intensity, size and shape between living T. vaginalis and other cells was also observed.

Our data are in accordance with Lopes et al. (2001), who showed, using specific monoclonal antibodies, that T. vaginalis presents a-tubulin at the flagella, basal bodies and the axostyle and b-tubulin at the axostyle and flagella, since FLUTAX-2 binds to ab-tubulin dimers. Although no positive results were found, it is possible that trichomonads present the g-tubulin isoform (Lopes et al. 2001).

Although 30236 and 30238 T. vaginalis strains present different sensitivities to metronidazole, our results demonstrated no differences in the binding profile of FLUTAX-2. The same was observed with the analyzed isolated strain, demonstrating that diagnosis of human trichomonosis with FLUTAX-2 may become a new alternative. Our new technique is still being tested on vaginal discharges and urine and must be improved for use as a routine laboratory method. Furthermore, an analysis of the composition of the cytoskeleton of T. vaginalis will contribute to the understanding of the biochemical aspects involved in cellular morphology and behavior, as well as the mechanisms related to host-parasite interactions.

 

ACKNOWLEDGEMENTS

To Dr Sérgio De Meda Lamb, Pharmacy School Chairman for his support; to Dr Denise C Machado, IPB, PUCRS, for microscope assistance and to Rafael V Michel and Fernanda S Meira for technical assistance.

 

REFERENCES

Benchimol M, Diniz JAP, Ribeiro K 2000. The fine structure of the axostyle and its associations with organelles in TrichomonadsTissue Cell 32: 178-187.

Borchardt KA, Smith RF 1991. An evaluation of an InPoucht TV culture method for diagnosingTrichomonas vaginalis infection. Genitourin Med 67: 149-152.

De Carli GA 2001. Exame de outros espécimes do trato intestinal e sistema urogenital. In GA De Carli, Parasitologia Clínica. Seleção de Métodos e Técnicas de Laboratório para o Diagnóstico das Parasitoses Humanas, Atheneu, Rio de Janeiro. p. 165-206.

DeMeo LR, Draper DL, McGregor JA, Moore DF, Peter CR, Kapernick OS, McCormack WM 1996. Evaluation of a deoxyribonucleic acid probe for the detection of Trichomonas vaginalis in vaginal secretions. Am J Obstet Gynecol 174: 1339-1342.

Diamond LS 1957. The establishment of various trichomonads of animals and man in axenic cultures. J Parasitol 43: 488-490.

Diaz JF, Strobe R, Engelborghs Y, Souto AA, Andreu JM 2000. Molecular recognition of taxol by microtubules: kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site. J Biol Chem 275: 26265-26276.

Fleming DT, Wasserheit JN 1999. From epidemiological synergy to public health policy and practice: the contribuition of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Inf 75: 3-17.

Gerbase AC, Rowley JT, Heyman HL, Berkley SFB, Piot P 1998. Global prevalence and incidence estimates of selected curable STDs. Sex Trans Inf 74: S12-S16.

Gram IT, Macaluso M, Churchill J, Stalsberg H 1992. Trichomonas vaginalis (TV) and human papillomavirus (HPV) infection and the incidence of cervical intraepithelial neoplasia (CIN) grade III.Cancer Causes Control 3: 231-236.

Grodstein F, Goldman MB, Cramer DW 1993. Relation of tubal infertility to history of sexually transmitted diseases. Am J Epidemiol 137: 577-584.

Heine P, McGregor JA 1993. Trichomonas vaginalis: a reemerging pathogen. Clin Obstet Gynecol 36: 137-144.

Heine RP, Wiesenfeld HC, Sweet RL, Witkin SS 1997. Polymerase chain reaction analysis of distal vaginal specimens: a less invasive strategy for detection of Trichomonas vaginalisClin Infect Dis 24: 985-987.

Honigberg BM, Burgess E 1994. Trichomonads of importance in human medicine includingDientamoeba fragilis. In JP Kreier, Parasitic Protozoa, 2 ed., v.9, Academic Press, San Diego, p. 1-57.

Kharsany AB, Hoosen AA, Moodley J, Bagaratee J, Gouws E 1993. The association between sexually transmitted pathogens and cervical intraepithelial neoplasia in a developing community.Genitourin Med 69: 357-360.

Krieger JN, Tam MR, Stevens CE, Nielsen IO, Hale J, Kiviat NB, Holmes KK 1988. Diagnosis of trichomonosis. Comparison of convencional wet-mount examination with cytologic studies, cultures, and monoclonal antibody staining of direct especimens. JAMA 259: 1223-1227.

Lehker MW, Alderete JF 2000. Biology of trichomonosis. Curr Opin Infect Dis 13: 37-45.

Lopes LC, Benchimol M, Ribeiro KC 2001. Immunolocalization of tubulin isoforms and post-translational modifications in the protist Tritrichomonas foetus and Trichomonas vaginalisHistochem Cell 116: 17-29.

Madico G, Quinn TC, Rompalo A, McKee Jr KT, Gaydos CA 1998. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples. J Clin Microbiol 36: 3205-3210.

Ohlemeyer CL, Homberger LL, Lynch DA, Swierkosz EM 1998. Diagnosis of Trichomonas vaginalisin adolescent females: InPoucht TV culture versus wet-mount microsopy. J Adolesc Health 22: 205-208.

Ribeiro KC, Monteiro-Leal LH, Benchimol M 2000. Contribution of the axostyle and flagella to the closed mitosis of Tritrichomonas foetus and Trichomonas vaginalisJ Eukaryot Microbiol 47: 481-492.

Shaio MF, Lin PR, Liu JY 1997. Colorimetric one-tube nested PCR for detection of Trichomonas vaginalis in vaginal discharge. J Clin Microbiol 35: 132-138.

Sharma P, Malla N, Gupta I, Ganguly NK, Mahajan RC 1991. A comparison of wet-mount, culture and enzyme im-munosorbent assay for the diagnosis of trichomonosis in women. Trop Geogr Med 43: 257-260.

Sorvillo F, Kerndt P 1998. Trichomonas vaginalis and amplification of HIV-1 transmission. Lancet 351: 213-214.

Souto AA, Acuña AU, Andreu JM, Barasoain I, Abal M, Amat-Guerri F 1995. New fluorescent water-soluble taxol derivatives. Angew Chem Int Ed Engl 34: 2710-2712.

Zhang ZF, Begg CB 1994. Is Trichomonas vaginalis a cause of cervical neoplasia? Results from a combinet analysis of 24 studies. Int J Epidemiol 23: 682-690.

+Corresponding author. Fax: +55-51-3332.2582. E-mail:  This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
Received 20 August 2002
Accepted 9 January 2003

This work was supported by Faculdade de Farmácia, PUCRS, and CNPq (# 470148/01-2). SBL is recipient of a fellowship (# 00501323) from Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul, and TT from the Conselho Nacional de Desenvolvimento Científico e Tecnológico.

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