• View in gallery

    Phylogenetic tree of 20 Trichomonas vaginalis reference isolates and 32 T. vaginalis clinical isolates constructed on the basis of the presence or absence of 33 individual Hsp70-RFLP bands using the phylogeny inference package FreeTree. 14 Numbers at the upper left of each node show the operational taxonomic unit (OUT) jackknifing percent values based on 10,000 resampled datasets, which estimate statistical support for the existence of those branches. The sum of the horizontal branch lengths between individual T. vaginalis isolates reflects the relative genetic distance separating the isolates. Boxed isolates were determined to be metronidazole resistant. Isolates marked with an asterisk (*) were determined to be metronidazole susceptible. Isolates marked with a raised pound symbol (#) were not tested for metronidazole susceptibility, but are presumed to be sensitive because patients responded to therapy and cleared the infection. Isolates without notation were not tested for metronidazole susceptibility.

  • 1.

    World Health Organization, 2001. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. Geneva: World Health Organization.

    • Search Google Scholar
    • Export Citation
  • 2.

    Cotch MF, Pastorek JG 2nd, Nugent RP, Hillier SL, Gibbs RS, Martin DH, Eschenbach DA, Edelman R, Carey JC, Regan JA, Krohn MA, Klebanoff MA, Rao AV, Rhoads GG, 1997. Trichomonas vaginalis associated with low birth weight and preterm delivery. Sex Transm Dis 24: 353360.

    • Search Google Scholar
    • Export Citation
  • 3.

    Minkoff H, Grunebaum AN, Schwarz RH, Feldman J, Cummings M, Crombleholme W, Clark L, Pringle G, McCormack WM, 1984. Risk factors for prematurity and premature rupture of membranes: a prospective study of the vaginal flora in pregnancy. Am J Obstet Gynecol 150: 965972.

    • Search Google Scholar
    • Export Citation
  • 4.

    McClelland RS, Sangare L, Hassan WM, Lavreys L, Mandaliya K, Kiarie J, Ndinya-Achola J, Jaoko W, Baeten JM, 2007. Infection with Trichomonas vaginalis increases the risk of HIV acquisition. J Infect Dis 195: 698702.

    • Search Google Scholar
    • Export Citation
  • 5.

    Sorvillo F, Smith L, Kerndt P, Ash L, 2001. Trichomonas vaginalis, HIV, and African-Americans. Emerg Infect Dis 7: 927932.

  • 6.

    Zhang ZF, Begg CB, 1994. Is Trichomonas vaginalis a cause of cervical neoplasia? Results from a combined analysis of 24 studies. Int J Epidemiol 23: 682690.

    • Search Google Scholar
    • Export Citation
  • 7.

    Stark JR, Judson G, Alderete JF, Mundodi V, Kucknoor AS, Giovannucci EL, Platz EA, Sutcliffe S, Fall K, Kurth T, Ma J, Stampfer MJ, Mucci LA, 2009. Prospective study of Trichomonas vaginalis infection and prostate cancer incidence and mortality: physicians' Health Study. J Natl Cancer Inst 101: 14061411.

    • Search Google Scholar
    • Export Citation
  • 8.

    Schmid G, Narcisi E, Mosure D, Secor WE, Higgins J, Moreno H, 2001. Prevalence of metronidazole-resistant Trichomonas vaginalis in a gynecology clinic. J Reprod Med 46: 545549.

    • Search Google Scholar
    • Export Citation
  • 9.

    Narcisi EM, Secor WE, 1996. In vitro effect of tinidazole and furazolidone on metronidazole-resistant Trichomonas vaginalis. Antimicrob Agents Chemother 40: 11211125.

    • Search Google Scholar
    • Export Citation
  • 10.

    Upcroft P, Upcroft JA, 2001. Drug targets and mechanisms of resistance in the anaerobic protozoa. Clin Microbiol Rev 14: 150164.

  • 11.

    Dunne RL, Dunn LA, Upcroft P, O'Donoghue PJ, Upcroft JA, 2003. Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis. Cell Res 13: 239249.

    • Search Google Scholar
    • Export Citation
  • 12.

    Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, Zhao Q, Wortman JR, Bidwell SL, Alsmark UC, Besteiro S, Sicheritz-Ponten T, Noel CJ, Dacks JB, Foster PG, Simillion C, Van de Peer Y, Miranda-Saavedra D, Barton GJ, Westrop GD, Müller S, Dessi D, Fiori PL, Ren Q, Paulsen I, Zhang H, Bastida-Corcuera FD, Simoes-Barbosa A, Brown MT, Hayes RD, Mukherjee M, Okumura CY, Schneider R, Smith AJ, Vanacova S, Villalvazo M, Haas BJ, Pertea M, Feldblyum TV, Utterback TR, Shu CL, Osoegawa K, de Jong PJ, Hrdy I, Horvathova L, Zubacova Z, Dolezal P, Malik SB, Logsdon JM Jr, Henze K, Gupta A, Wang CC, Dunne RL, Upcroft JA, Upcroft P, White O, Salzberg SL, Tang P, Chiu CH, Lee YS, Embley TM, Coombs GH, Mottram JC, Tachezy J, Fraser-Liggett CM, Johnson PJ, 2007. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315: 207212.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lossick JG, Muller M, Gorrell TE, 1986. In vitro drug susceptibility and doses of metronidazole required for cure in cases of refractory vaginal trichomoniasis. J Infect Dis 153: 948955.

    • Search Google Scholar
    • Export Citation
  • 14.

    Meade JC, de Mestral J, Stiles JK, Secor WE, Finley RW, Cleary JD, Lushbaugh WB, 2009. Genetic diversity of Trichomonas vaginalis clinical isolates determined by EcoRI restriction fragment length polymorphism of heat-shock protein 70 genes. Am J Trop Med Hyg 80: 245251.

    • Search Google Scholar
    • Export Citation
  • 15.

    Hampl V, Pavlíček A, Flegr J, 2001. Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites. Int J Syst Evol Microbiol 51: 731735.

    • Search Google Scholar
    • Export Citation
  • 16.

    Saitou N, Mei M, 1987. The neighbor-joining method: a new method for reconstruction of phylogenetic trees. Mol Biol Evol 4: 406425.

  • 17.

    Nei M, Li W-H, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci U S A 76: 52695273.

    • Search Google Scholar
    • Export Citation
  • 18.

    Campos Aldrete ME, Salgado-Zamora H, Luna J, Meléndez E, Meráz-Ríos MA, 2005. A high-throughput colorimetric and fluorometric microassay for the evaluation of nitroimidazole derivatives anti-Trichomonas activity. Toxicol In Vitro 19: 10451050.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Genetic Relatedness of Trichomonas vaginalis Reference and Clinical Isolates

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  • Department of Microbiology and Department of Medicine, Division of Infectious Diseases, University of Mississippi Medical Center, Jackson, Mississippi

We have determined the metronidazole susceptibility status of 20 Trichomonas vaginalis isolates from American Type Culture Collection (ATCC) and assessed the level of genetic relatedness in these isolates using 32 additional T. vaginalis clinical isolates for comparison. These ATCC isolates are commonly used as reference strains in T. vaginalis research and this information provides a rational basis for selection of reference strains for use in comparative studies of T. vaginalis phenotypic and clinical characteristics.

Trichomonas vaginalis is a protozoan parasite that infects the urogenital tract of humans, causing trichomoniasis, the most common non-viral, sexually transmitted disease in the world. Symptoms of T. vaginalis infection include vaginitis and cervicitis in women and urethritis in men, although asymptomatic infection does occur. 1 The prevalence of T. vaginalis infection is significantly underestimated because of the frequency of asymptomatic infections. Trichomoniasis is associated with a higher risk for other infectious diseases and adverse pregnancy outcomes such as preterm birth, premature rupture of placental membranes, and low birth weight infants. 2, 3 Trichomonas vaginalis infection increases the risk of human immunodeficiency virus (HIV) acquisition and the Centers for Disease Control and Prevention (CDC) estimates that as much as 20% of HIV transmission in the African American population is attributable to T. vaginalis infection. 4, 5 Trichomonas vaginalis infection also increases the risk of cervical neoplasia and prostate cancer. 6, 7 Exposure to T. vaginalis results in a 2-fold increase in the risk of diagnosis of extra-prostatic prostate cancer and a 3-fold increase in the risk of cancer that led to cancer-specific death. 7 Thus, although trichomoniasis itself is a curable disease, T. vaginalis infection may indirectly be a life threatening disease. The primary treatment of T. vaginalis infection, metronidazole, a heterocyclic 5-nitroimidazole, is usually very effective and well tolerated. However, resistance is becoming increasingly widespread and it is estimated that 2.5–10% of all cases of trichomoniasis display some degree of resistance to initial treatment with metronidazole. 811

Trichomonas vaginalis infection is highly variable in its clinical manifestations such as pathogenicity, metronidazole susceptibility, and patient susceptibility to acquisition of other infectious agents. These differences may be associated with genotypic strain variation in T. vaginalis as is the case with many infectious agents. The recent advent of proteomic and genomic techniques based on the T. vaginalis genome sequence presents an opportunity to elucidate the genetic factors controlling clinical manifestations of trichomoniasis. 12 Over time, many clinical strains of T. vaginalis have been deposited into the American Type Culture Collection (ATCC, Manassas, VA) repository. These strains are often used as reference strains when characterizing current clinical isolates and in molecular studies of T. vaginalis.

We have determined the metronidazole susceptibility and genotype of the 20 historic isolates from the ATCC repository that are most frequently used in T. vaginalis research. Collectively, these isolates have been referenced 133 times according to the ATCC website (www.ATCC.org), with one isolate (30001) referenced 47 times. We have also found many additional literature references for these isolates that were not cited on the ATCC website. The ATCC isolates tested are listed in Table 1 with the year of initial culture, their common strain designation, and geographic origin. These reference strains come from nine different states, two foreign locations, and the dates of sample collection range from 1939 to 1986. Strain Pra98, common designation G3, is the isolate used to determine the T. vaginalis genome sequence. 12

Table 1

Trichomonas vaginalis reference and clinical isolates with geographic origin, date of original culture, and metronidazole susceptibility

IsolateATCCLocationYearMIC (µg/mL) *
Number
P 1Jackson, MS19951000
MSA 806Akron, OH1995> 400
MSA 812Birmingham, AL1996> 400
MSA 836Greenville, NC1997> 400
MSA 844Rochester, NY1998> 400
MSA 809BBrooklyn, NY1996400
MSA 811Benton Harbor, MI1996400
RU 35750139PA1982200
RU 38250141CT1983200
CDC 08550143Columbus, OH1980150
RU 38450140MA1983125
RU 39350142NY1983125
GMH 29Atlanta, GA199775
IR 7850138Vienna, Austria197862
JH 32A #430238Baltimore, MD196362
E 44Jackson, MS200132
GMH 47Atlanta, GA199725
GMH 67Atlanta, GA199712.5
C-1:NIH30001U19568
HsD:NIH50183MD19648
W 096Jackson, MS20098
B 95Jackson, MS1993< 8
B 110Jackson, MS1993< 8
B 137Jackson, MS1993< 8
E 34Jackson, MS2001< 8
NYH 28650148New York, NY19774
W 019Jackson, MS20094
W 052Jackson, MS20094
W 075Jackson, MS20094
1176930092Denver, CO19582
B7RC250167Greenville, NC19862
TV 130247U19392
12341330186Nashville, TN19641
165307-130187Denver, CO19621
CDC 33750144Columbus, OH19831
G3Pra98United Kingdom19731
RP30188United KingdomU1
TVC30245Northampton, MA19661
TVC130246Bethesda, MD19560.5
#213Jackson, MS1991NT#
#294Jackson, MS1991NT#
C 6Jackson, MS1993NT#
C 11Jackson, MS1993NT#
C 14Jackson, MS1993NT#
S 1Jackson, MS1995NT#
S 3Jackson, MS1998NT#
T 1MJackson, MS2000NT#
E 70Jackson, MS2001NT
E 50Jackson, MS2001NT
E 95Jackson, MS2001NT
E56Jackson, MS2001NT
E 88Jackson, MS2001NT

Minimum inhibitory concentration (MIC) in µg/mL: high metronidazole resistance is an MIC ≥ 100 µg/mL, moderate metronidazole resistance is an MIC of 10–100 µg/mL, and metronidazole susceptibility is an MIC < 10 µg/mL.

= Data from prior study. 13

ATCC = American Type Culture Collection; U = data unknown; NT = not tested; NT# = not tested but presumed sensitive as patients responded to therapy and cleared infection.

Restriction fragment length polymorphism (RFLP) analysis of the ATCC isolates using a multilocus heat-inducible cytoplasmic heat-shock protein 70 (Hsp70) hybridization probe with EcoRI-digested genomic DNA was used in molecular typing of T. vaginalis isolates as described. 14 A phylogenetic tree assessing the relatedness of the T. vaginalis isolates was created by analyzing their Hsp70 RFLP patterns with Free Tree software using the Nei-Li coefficient of similarity and neighbor-joining method for tree construction. 1517 Thirty-two additional T. vaginalis clinical isolates, 23 collected in Jackson, MS between 1993 and 2001 and nine isolates from six states (AL, GA, MI, NY, OH, SC) collected between 1996 and 1998 were included in the phylogenetic analysis. Metronidazole susceptibility of the ATCC isolates was assessed using a modified Alamar Blue colorimetric microassay. 18 Minimal inhibitory concentration (MIC) was determined as the lowest concentration at which trophozoites were not motile.

In the 52 isolates typed by Hsp70 RFLP of EcoRI-digested genomic DNA, 7–11 distinct DNA bands, ranging in size from 1.8 to 22.5 kb, were detected. A total of 33 different DNA bands were identified in this analysis; 23 of those bands are present in these ATCC reference strains. Two ATCC isolates contained 10 bands, nine isolates contained 9 bands, eight isolates contained 8 bands, and one isolate contained 7 bands. The 5.5, 3.7, and 2.7 kb bands were present in all isolates, the 5.8 kb band was present in 19 ATCC isolates, the 4.4 kb band was present in 18 isolates and five bands were present in single ATCC isolates. The Hsp70 patterns are reproducible because several isolates were run multiple times yielding an identical RFLP pattern after every run. The RFLP patterns did not change over time because identical patterns were observed for DNA samples obtained from aliquots of T. vaginalis isolates cryopreserved in different years. A large degree of diversity was observed among the ATCC isolates as the 52 isolates, ATCC and clinical, produced 49 distinct RFLP banding patterns. There were three instances of identical Hsp70 RFLP patterns obtained from ATCC isolates and T. vaginalis clinical isolates widely separated in time of collection and geographic origin: ATCC 50139 (PA, 1982) and MSA806 (Akron, OH, 1995), ATCC 30247 (location unknown, 1939) and GMH 47 (Atlanta, GA, 1997), and ATCC 50140 (MA, 1983) and E44 (Jackson, MS, 2001). The existence of isolates indistinguishable by the HSP70 RFLP technique could indicate the widespread dispersion of individual isolates and their temporal stability over at least 58 years (1939 to 1997) in the United States. However, the presence of matching patterns could also be attributed to monomorphism among the T. vaginalis isolates.

The phylogenetic analysis indicates the possibility of two distinct lineages of T. vaginalis strains, Group I (upper group) with 31 strains and Group II (lower group) containing 21 strains ( Figure 1), which are characterized by deeply diverging branches containing individual T. vaginalis strains. The T. vaginalis isolates are distributed throughout the phylogenetic tree without regard for geographical origin or date of original culture ( Figure 1 and Table 1). There is significant statistical support for the placement of the isolates within the phylogram as indicated by the operational taxonomic unit-based jackknifing values calculated for each node. This is especially evident at the distal nodes ( Figure 1). Metronidazole-resistant isolates are boxed in Figure 1. Although 12 of 18 resistant isolates are present in the smaller Group II, this may be a reflection of the increased percentage of national isolates in Group II, 14 of 21, as compared with Group I where only 15 of 31 isolates came from outside Mississippi.

Figure 1.
Figure 1.

Phylogenetic tree of 20 Trichomonas vaginalis reference isolates and 32 T. vaginalis clinical isolates constructed on the basis of the presence or absence of 33 individual Hsp70-RFLP bands using the phylogeny inference package FreeTree. 14 Numbers at the upper left of each node show the operational taxonomic unit (OUT) jackknifing percent values based on 10,000 resampled datasets, which estimate statistical support for the existence of those branches. The sum of the horizontal branch lengths between individual T. vaginalis isolates reflects the relative genetic distance separating the isolates. Boxed isolates were determined to be metronidazole resistant. Isolates marked with an asterisk (*) were determined to be metronidazole susceptible. Isolates marked with a raised pound symbol (#) were not tested for metronidazole susceptibility, but are presumed to be sensitive because patients responded to therapy and cleared the infection. Isolates without notation were not tested for metronidazole susceptibility.

Citation: The American Society of Tropical Medicine and Hygiene 83, 6; 10.4269/ajtmh.2010.09-0718

The degree of metronidazole resistance for an individual isolate can vary depending on the method used to test susceptibility, complicating comparisons of metronidazole susceptibility assessed in different laboratories. Therefore, we tested all strains using the same susceptibility method to provide more precise data for comparing clinical isolates in the future. Of the 20 ATCC T. vaginalis isolates tested, two were found to be moderately resistant, 30238 and 50138, and five isolates were highly resistant, 50139, 50140, 50141, 50142, and 50143 ( Table 1). This determination was consistent with prior studies, which reported ATCC strains 30001, 50144, 50148, and Pra98 as metronidazole sensitive and strains 30238, 50138, 50139, 50140, 50141, 50142, and 50143 as metronidazole resistant. No correlation between Hsp70 RFLP band pattern or the presence of individual bands and metronidazole resistance was observed. Resistance observed in isolates was also not associated with a particular geographic location. The prevalence of resistance found in this study (32%) is significantly higher than the national average of 2–5% reported by CDC. This is likely because many of these strains were submitted to ATCC and CDC as a result of their resistance to metronidazole. Therefore, the prevalence of metronidazole resistance present in this set of reference strains does not necessarily represent that of the general population.

With the increased interest in T. vaginalis epidemiology, and phenotypic and genetic variation, reference strains are used as the basis for much of the research conducted. The selection of T. vaginalis isolates as reference strains to characterize and compare current clinical isolates on the basis of phenotypic variation will need to consider their genetic similarity to those clinical isolates to maximize the discovery of relevant genetic elements controlling phenotype and avoid differences rooted in genetic diversity. This is the first publication to assess the level of genetic diversity in and metronidazole susceptibility status of 20 reference isolates of T. vaginalis. The additional information presented on these reference strains provides a more complete description of these strains that have been frequently used for comparison with recently isolated clinical strains. For laboratories that use the EcoRI Hsp70 RFLP method, this permits more precise comparisons between reference isolates and clinical isolates to help elucidate the basis for variation in clinical manifestations and epidemiology of T. vaginalis infections.

ACKNOWLEDGMENTS:

We thank Jinrong Wei, Christopher Richmond, and Sapna Naik for technical assistance.

  • 1.

    World Health Organization, 2001. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. Geneva: World Health Organization.

    • Search Google Scholar
    • Export Citation
  • 2.

    Cotch MF, Pastorek JG 2nd, Nugent RP, Hillier SL, Gibbs RS, Martin DH, Eschenbach DA, Edelman R, Carey JC, Regan JA, Krohn MA, Klebanoff MA, Rao AV, Rhoads GG, 1997. Trichomonas vaginalis associated with low birth weight and preterm delivery. Sex Transm Dis 24: 353360.

    • Search Google Scholar
    • Export Citation
  • 3.

    Minkoff H, Grunebaum AN, Schwarz RH, Feldman J, Cummings M, Crombleholme W, Clark L, Pringle G, McCormack WM, 1984. Risk factors for prematurity and premature rupture of membranes: a prospective study of the vaginal flora in pregnancy. Am J Obstet Gynecol 150: 965972.

    • Search Google Scholar
    • Export Citation
  • 4.

    McClelland RS, Sangare L, Hassan WM, Lavreys L, Mandaliya K, Kiarie J, Ndinya-Achola J, Jaoko W, Baeten JM, 2007. Infection with Trichomonas vaginalis increases the risk of HIV acquisition. J Infect Dis 195: 698702.

    • Search Google Scholar
    • Export Citation
  • 5.

    Sorvillo F, Smith L, Kerndt P, Ash L, 2001. Trichomonas vaginalis, HIV, and African-Americans. Emerg Infect Dis 7: 927932.

  • 6.

    Zhang ZF, Begg CB, 1994. Is Trichomonas vaginalis a cause of cervical neoplasia? Results from a combined analysis of 24 studies. Int J Epidemiol 23: 682690.

    • Search Google Scholar
    • Export Citation
  • 7.

    Stark JR, Judson G, Alderete JF, Mundodi V, Kucknoor AS, Giovannucci EL, Platz EA, Sutcliffe S, Fall K, Kurth T, Ma J, Stampfer MJ, Mucci LA, 2009. Prospective study of Trichomonas vaginalis infection and prostate cancer incidence and mortality: physicians' Health Study. J Natl Cancer Inst 101: 14061411.

    • Search Google Scholar
    • Export Citation
  • 8.

    Schmid G, Narcisi E, Mosure D, Secor WE, Higgins J, Moreno H, 2001. Prevalence of metronidazole-resistant Trichomonas vaginalis in a gynecology clinic. J Reprod Med 46: 545549.

    • Search Google Scholar
    • Export Citation
  • 9.

    Narcisi EM, Secor WE, 1996. In vitro effect of tinidazole and furazolidone on metronidazole-resistant Trichomonas vaginalis. Antimicrob Agents Chemother 40: 11211125.

    • Search Google Scholar
    • Export Citation
  • 10.

    Upcroft P, Upcroft JA, 2001. Drug targets and mechanisms of resistance in the anaerobic protozoa. Clin Microbiol Rev 14: 150164.

  • 11.

    Dunne RL, Dunn LA, Upcroft P, O'Donoghue PJ, Upcroft JA, 2003. Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis. Cell Res 13: 239249.

    • Search Google Scholar
    • Export Citation
  • 12.

    Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, Zhao Q, Wortman JR, Bidwell SL, Alsmark UC, Besteiro S, Sicheritz-Ponten T, Noel CJ, Dacks JB, Foster PG, Simillion C, Van de Peer Y, Miranda-Saavedra D, Barton GJ, Westrop GD, Müller S, Dessi D, Fiori PL, Ren Q, Paulsen I, Zhang H, Bastida-Corcuera FD, Simoes-Barbosa A, Brown MT, Hayes RD, Mukherjee M, Okumura CY, Schneider R, Smith AJ, Vanacova S, Villalvazo M, Haas BJ, Pertea M, Feldblyum TV, Utterback TR, Shu CL, Osoegawa K, de Jong PJ, Hrdy I, Horvathova L, Zubacova Z, Dolezal P, Malik SB, Logsdon JM Jr, Henze K, Gupta A, Wang CC, Dunne RL, Upcroft JA, Upcroft P, White O, Salzberg SL, Tang P, Chiu CH, Lee YS, Embley TM, Coombs GH, Mottram JC, Tachezy J, Fraser-Liggett CM, Johnson PJ, 2007. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315: 207212.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lossick JG, Muller M, Gorrell TE, 1986. In vitro drug susceptibility and doses of metronidazole required for cure in cases of refractory vaginal trichomoniasis. J Infect Dis 153: 948955.

    • Search Google Scholar
    • Export Citation
  • 14.

    Meade JC, de Mestral J, Stiles JK, Secor WE, Finley RW, Cleary JD, Lushbaugh WB, 2009. Genetic diversity of Trichomonas vaginalis clinical isolates determined by EcoRI restriction fragment length polymorphism of heat-shock protein 70 genes. Am J Trop Med Hyg 80: 245251.

    • Search Google Scholar
    • Export Citation
  • 15.

    Hampl V, Pavlíček A, Flegr J, 2001. Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with the freeware program FreeTree: application to trichomonad parasites. Int J Syst Evol Microbiol 51: 731735.

    • Search Google Scholar
    • Export Citation
  • 16.

    Saitou N, Mei M, 1987. The neighbor-joining method: a new method for reconstruction of phylogenetic trees. Mol Biol Evol 4: 406425.

  • 17.

    Nei M, Li W-H, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci U S A 76: 52695273.

    • Search Google Scholar
    • Export Citation
  • 18.

    Campos Aldrete ME, Salgado-Zamora H, Luna J, Meléndez E, Meráz-Ríos MA, 2005. A high-throughput colorimetric and fluorometric microassay for the evaluation of nitroimidazole derivatives anti-Trichomonas activity. Toxicol In Vitro 19: 10451050.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Denise C. Cornelius, Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505. E-mail: dcornelius@umc.edu

Financial support: This work was partially funded by the Mississippi Institute for Improvement of Geographic Minority Health.

Authors' addresses: Denise C. Cornelius, William B. Lushbaugh, and John C. Meade, Department of Microbiology, University of Mississippi Medical Center, Jackson, MS, E-mails: dcornelius@umc.edu, wlushbaugh@umc.edu, and jmeade@umc.edu. Leandro Mena, Department of Medicine, Division of Infectious Disease, University of Mississippi Medical Center, Jackson, MS, E-mail: lmena@umc.edu.

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