Detection and biovar discrimination of Ureaplasma urealyticum by real-time PCR

https://doi.org/10.1016/j.mcp.2005.04.002Get rights and content

Abstract

Prenatal intrauterine infection has been recognized as an important cause of premature birth, and Ureaplasma urealyticum is one of the commonest pathogens. U. urealyticum consists of 14 serovars that can be divided into two biovars (parvo and T960), and the pathogenicity of U. urealyticum may be different according to the biovar. To detect U. urealyticum and determine its biovar simultaneously, we developed a real-time polymerase chain reaction (PCR) assay targeting urease gene. The real-time PCR biovar-typed two reference strains and 42 culture isolates of U. urealyticum as correctly as conventional PCR with direct sequencing. Subsequently, 87 clinical specimens (amniotic fluid, cord blood, vaginal swab) were tested for culture, conventional PCR, and real-time PCR. When compared with conventional PCR, sensitivity and specificity of real-time PCR were 89.5 and 98.5%, respectively, and those of culture were 47.4 and 100%, respectively. Of 18 clinical specimens that were found positive and biovar-typed by real-time PCR, parvo biovar was 66.7% and T960 biovar was 33.3%. This real-time PCR assay can be useful for the simultaneous detection and biovar discrimination of U. urealyticum in clinical specimens. Further study to quantify U. urealyticum would be facilitated on the basis of this method.

Introduction

Premature birth is the most important cause of perinatal morbidity and mortality. Prenatal intrauterine infection has been implicated in the etiology of preterm birth, and Ureaplasma urealyticum is recognized as one of the most common pathogens. U. urealyticum is the microorganism most frequently isolated from the amniotic fluid in women with term and preterm labor and with premature rupture of membrane [1], [2], [3]. U. urealyticum is not only a commensal of lower genital tract in 40–80% of sexually active women [4], but also is associated with chorioamnionitis, spontaneous abortion, perinatal morbidities and mortalities [4], [5], [6], [7].

U. urealyticum consists of 14 serovars that can be divided into two biovars, parvo and T960, on the basis of phenotypic and genotypic characteristics. The ‘parvo biovar’ comprises four serovars (serovars 1, 3, 6, 14), while the ‘T960 biovar’ includes 10 serovars (serovars 2, 4, 5, 7–13) [8], [9], [10], [11], [12], [13], [14]. Although many genes of U. urealyticum show high degree of homology, the sequences of some genes are so divergent between the two biovars. Randomly amplified polymorphic DNA tests or polymerase chain reaction (PCR) of 16S rRNA, 16S-23S rRNA intergenic region, genus-defining urease, serovar-defining multiple-banded antigen (MBA) genes may be used to clearly distinguish between the biovars. Based on these findings, it has recently been proposed that the parvo biovar should be designated as a separate species, U. parvum [15].

The diversity between the two biovars may be responsible for the differences in pathogenicity, cultural fastidiousness, or response to treatment. Several studies [7], [16], [17], [18], [19] have demonstrated that certain serovars are associated with invasive disease and that T960 biovar is more often associated with adverse pregnancy outcomes. However, in some studies [4], [20], [21], [22], [23], no correlation has been found between serovars and pathogenicity.

As standard cultures of U. urealyticum are difficult, time-consuming, and of low sensitivity, their clinical utility is limited. Thus, it is crucial to detect U. urealyticum more accurately, rapidly, and sensitively than standard culture methods. It is also necessary to establish a method that can discriminate between biovars easily. The aim of this study was to develop a real-time PCR assay that would allow simultaneous detection and biovar-typing of U. urealyticum in clinical specimens.

Section snippets

Study subjects

Two reference strains of U. urealyticum, ATCC 27815 (serovar 3, the type strain of parvo biovar) and ATCC 27618 (serovar 8, the type strain of T960 biovar), were obtained from American Type Culture Collection (Manassas, VA). Forty-two strains of U. urealyticum were isolated by broth culture method (MYCOFAST EVOLUTION 2, International Microbio, Signes, France) from clinical specimens in Seoul National University Hospital. These reference strains and culture isolates were tested for conventional

Results

The reference strains and 42 culture isolates of U. urealyticum all produced specific 378 base-pair amplicons from the conventional PCR (Fig. 1). Direct sequencing analysis of the amplicons identified each reference strain correctly as the corresponding biovar (i.e. ATCC 27815 as parvo and ATCC 27618 as T960). The results of direct sequencing of 42 culture isolates were as follows: 37 (88.1%) were parvo biovars, 4 (9.5%) were T960 biovars, and one (2.4%) specimen contained both biovars. The

Discussion

In this study U. urealyticum was detected in 54.8% of vaginal swabs by conventional PCR, which consists with the results of previous studies [4]. The number of tested specimens being small, positive rates in amniotic fluid and cord blood specimens could not accurately reflect the true positive rates in those specimens. Considering 40–80% of sexually active women have U. urealyticum in their lower genital tracts, however, it would be deduced that ascending infection caused by U. urealyticum

Acknowledgements

This study was supported by grant 01-PJ1-PG1-01CH07-0002 from the 2004 Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea. We thank Eun-Jung Lee and Se-Ik Joo for technical assistance.

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