Detection and biovar discrimination of Ureaplasma urealyticum by real-time PCR
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.
References (31)
- et al.
Clinical implications of detection of Ureaplasma urealyticum in the amniotic cavity with the polymerase chain reaction
Am J Obstet Gynecol
(2000) - et al.
Serologic evidence of Ureaplasma urealyticum infection in women with spontaneous pregnancy loss
Am J Obstet Gynecol
(1983) - et al.
Arbitrarily-primed PCR confirms the differentiation of strains of Ureaplasma urealyticum into two biovars
Mol Cell Probes
(1995) - et al.
Problems associated with serotyping strains of Ureaplasma urealyticum
Dian Microbiol Infect Dis
(1985) Rapid allelic discrimination from real-time DNA amplification
Methods
(2001)- et al.
The clinical significance of detecting Ureaplasma urealyticum by the polymerase chain reaction in the amniotic fluid of patients with preterm labor
Am J Obstet Gynecol
(2003) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream
Exp Hematol
(2002)- et al.
Microbial invasion of the amniotic cavity during term labor. Prevalence and clinical significance
J Reprod Med
(1993) - et al.
The association of occult amniotic fluid infection with gestational age and neonatal outcome among women in preterm labor
Obstet Gynecol
(1992) - et al.
Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns
Clin Microbiol Rev
(1993)
A case-control study of chorioamnionic infection and histologic chorioamnionitis in prematurity
N Engl J Med
Association of Ureaplasma urealyticum in the placenta with perinatal morbidity and mortality
N Engl J Med
Detection of Ureaplasma urealyticum by PCR and biovar determination by liquid hybridization
J Clin Microbiol
Biovar diversity is reflected by variations of genes encoding urease of Ureaplasma urealyticum
Microbiol Immunol
Ureaplasma urealyticum biovar specificity and diversity are encoded in multiple-banded antigen gene
J Clin Microbiol
Cited by (72)
Mycoplasmas and ureaplasmas
2023, Molecular Medical Microbiology, Third EditionClinical performance of four multiplex real-time PCR kits detecting urogenital and sexually transmitted pathogens
2022, Clinical Microbiology and InfectionCitation Excerpt :The performance of the kits for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis was compared to that of the cobas CT/NG and TV/MG kits (Roche Diagnostics, USA) [5–10]. For the two kits that also detect Ureaplasma urealyticum, U. parvum and Mycoplasma hominis, the detection performance was compared to that of published in-house TaqMan PCRs [11,12]. Between March and October 2019, remnants of clinical samples in cobas PCR medium (Roche Molecular Systems) received at the French National Reference Centre for Bacterial STIs in the Bacteriology Department of Saint-Louis Hospital (Paris, France) were prospectively and consecutively collected and stored at –80°C until testing with commercial kits.
A specific bacterial DNA signature in the vagina of Australian women in midpregnancy predicts high risk of spontaneous preterm birth (the Predict1000 study)
2021, American Journal of Obstetrics and GynecologyCitation Excerpt :All reactions were conducted on a ViiA7 real-time PCR system and data were analyzed using QuantStudio Real-Time PCR Software v1.3 (Life Technologies, Carlsbad, CA). U parvum and U urealyticum DNA was detected from UTM vaginal swab DNA using real-time PCR targeting the urease gene, as described by Yi et al.30 Reaction mixtures (final concentration) consisted of 1X Taqman FAST Advanced Master Mix (Life Technologies), 0.9 μM primers UU1613F and UU1524R (Life Technologies), 0.25 μM probes UU-parvo-FAM and UU-T960-VIC (Life Technologies), 5 μL of template DNA, and nuclease-free water (Integrated DNA Technologies) to a final volume of 20 μL. PCR cycling conditions consisted of an initial denaturation or Taq activation at 95oC for 20 seconds, followed by 40 quantification cycles of 95°C for 1 second and 60°C for 20 seconds (data acquiring).
Ureaplasma parvum causes hyperammonemia presenting as refractory status epilepticus after kidney transplant
2020, Journal of Critical CareA case of Ureaplasma parvum meningitis in an adult after transphenoidal ablation of craniopharyngioma
2019, International Journal of Infectious DiseasesCitation Excerpt :The 842 bp sequencing product was compared with sequences deposited in the GenBank database and results revealed 99.8% identity with U. parvum isolates, and 98.7–99.8% identity with U. urealyticum isolates. This result was confirmed by PCR analysis for U. urealyticum and U. parvum by the French National Reference Center for bacterial STIs, which identified U. parvum in the CSF sample (Yi et al., 2005). Levofloxacin (1 g daily PO) was maintained for 3 weeks.
Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis
2019, Journal of Microbiology, Immunology and Infection