Typing of Chlamydia trachomatis strains from urine samples by amplification and sequencing the major outer membrane protein gene (omp1)

Objectives: To develop a novel protocol for the extraction, amplification, and sequencing of Chlamydia trachomatis MOMP gene (omp1) from urine, a non- invasive source, and apply it to an epidemiological study on the distributi on of C trachomatis strains in a population of pregnant women in Thailand. Methods: The C trachomatis DNA was extracted from culture stocks and urine using a slightly modified commercially available kit, the High Pure PCR Tem plate Preparation Kit (Roche Molecular Biochemicals, IN, USA). The PCR and sequencing primers used for the amplification and sequencing of the omp1 we re designed based on the nucleotide sequence of multiple C trachomatis stra ins found in GenBank. The protocol for the extraction, amplification, and s equencing was tested on laboratory culture stocks of reference strains of a ll C trachomatis serovars and on urine samples collected in a cross section al study designed to assess the prevalence of C trachomatis infections in t he cities of Bangkok and Chiang Rai, Thailand. Results: The omp1 gene was successfully amplified and sequenced from 18 lab oratory C trachomatis reference strains and from 45 C trachomatis positive urine clinical samples collected from asymptomatic pregnant women. Among cl inical samples, we found nine different C trachomatis genotypes: F (11, 25% ), D (10, 22.6%), H (5, 11.7%), K (5, 11.7%), E (4, 9.3,%), la (3, 7%), B ( 3, 7%), Ja (2, 4.5%), and G (1, 2.3%). One specimen generated an omp1 DNA s equence pattern indicating the presence of a mixed infection with at least two different serovars. Conclusions: Urine is a convenient and reliable source for genotyping C tra chomatis strains. A clear advantage of urine over traditional samples, such as cervical swabs, is that urine is a noninvasive source which makes colle ction easier and thus facilitates the enrolment of patients in clinical and epidemiological studies. In addition to typing, urine is increasingly used for diagnosis of C trachomatis infection by several commercially available nucleic acid amplification assays which represents a distinct advantage fo r collecting, transport, storage, and laboratory handling of samples.