Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR–restriction fragment length polymorphism

Abstract

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR-restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI. It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.

Genetic Characterization of Trichomonas vaginalis Isolates by Use of Multilocus Sequence Typing

“…Bootstrap support for the two clades in P1, P8, and P13 was negligible (Ͻ40%). P8 contained the greatest number of alleles (17) and displayed an increased number of small groups of isolates. The two major clades identified by the individual trees had similar distributions of STs into clades with particular STs clustering together in a single clade.…”

“…Currently, there is no "gold standard" method for genotyping isolates of T. vaginalis. A number of methods have been used in previous attempts to characterize the genetic diversity and population structure of T. vaginalis including isoenzyme analysis, repetitive sequence hybridization, random amplified polymorphic DNA, restriction fragment length polymorphism (RFLP), sequence polymorphisms in rRNA genes and intergenic regions, pulsed-field gel electrophoresis, and antigenic characterization (3,17,26,43,49,50,58,60,64,65). These methods have variable levels of reproducibility and precision in determining the genetic relatedness of isolates.…”

Determination of Trichomonas vaginalis Genotypes Using PCR-Restriction Fragment Length Polymorphism (RFLP)

“…Bu çalışmalarda PZR-Tek Zincir Konformosyonel Polimorfizm (PZR-SSCP), PZR-Restriksiyon Parça Uzunluk Polimorfizmi (RFLP), Çoklu Lokus Sekans Tiplendirme (MLST), mikrosatellit analizi gibi yöntemler kullanılmıştır (10)(11)(12). Bu yöntemlerin büyük çoğunluğu ribozomal ve interjenik bölgelerdeki sekans polimorfizmlerinin belirlenmesi esasına dayanmaktadır (13).…”

“…Moleküler tiplendirme çalışmaları ile belirli topluluklarda T. vaginalis'in genetik çeşitliliği, popülasyon yapısı ve epidemiyolojik bağlan-tıları hakkında bilgi sahibi olunabilmektedir (18,19). Ayrıca, genetik çeşitliliğinin araştırılması parazitin patojenitesini, TVV ilişkisini, ilaç direncini ve tekrarlayan enfeksiyonların kaynağını belirlemeye bü-yük katkı sağlayacağı düşünülmektedir (10,20,21). Çalışmamızda, T. vaginalis izolatlarının genotipleri aktin geninin RFLP paternine göre belirlenmiştir.…”

“…Referans olarak ele aldığımız Cirucitti ve ark. bildirdiği yöntemden farklı olarak çalışmamızda PZR ürünleri kesim öncesi saflaştırılmıştır (10). Mevcut yöntem uygulandığında agaroz jelde kesim ürünleri zayıf görülürken saflaştırma sonrası ürün yoğun-luğunun artması sonucu daha parlak bantlar elde edilmiştir.…”