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Ureaplasma Urealyticum and Ureaplasma Parvum

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Ureaplasma Urealyticum and Ureaplasma Parvum The Journal of Molecular Diagnostics, Vol. 13, No. 2, March 2011 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.jmoldx.2010.10.007 Modified Real-Time PCR for Detecting, Differentiating, and Quantifying Ureaplasma urealyticum and Ureaplasma parvum Ellen Vancutsem,* Oriane Soetens,* microbiological methods and are, therefore, usually re- Maria Breugelmans,† Walter Foulon,† ferred to as Ureaplasma species. They are found in the and Anne Naessens* lower genital tract of nearly 50% of pregnant women as part of the normal vaginal flora. However, in some cases, From the Departments of Microbiology and Infection Control* Ureaplasma species have interfered with normal fetal de- and Obstetrics,† Universitair Ziekenhuis Brussel, Brussels, velopment by causing an ascending infection.2–7 The Belgium reason for this infection is not fully understood but may be associated with the virulence of the microorganism, the host immune system, or local factors present in the lower We evaluated a previously described quantitative real- genital tract. Species differentiation might be important time PCR (qPCR) for quantifying and differentiating because previous studies2,8,9 suggest that nongonococ- Ureaplasma parvum and U. urealyticum. Because of cal urethritis and an adverse pregnancy outcome with nonspecific reactions with Staphylococcus aureus respect to birth weight, gestational age, and preterm DNA in the U. parvum PCR, we developed a modified delivery are implicated with the presence of U. urealyti- qPCR and designed new primers. These oligonucleo- cum and not with U. parvum. tides eradicated cross-reactions, indicating higher Because strains can only be differentiated with la- specificity. The detection limits of the qPCR were de- bor-intensive serotyping, an easy-to-perform sensitive termined at 1 and 3 colony-forming units/ml for U. method for differentiating the two species is necessary on parvum and U. urealyticum, respectively. The quan- initiation of pathogenicity studies. Quantitative real-time tification limits of the assay for both Ureaplasma PCR (qPCR) presents an interesting option because the 6 1 species ranged from 2.10 to 2.10 copy numbers per strains can be differentiated together, with evaluation of PCR. A total of 300 patient samples obtained from the the microbial burden in the lower genital tract. Moreover, lower genital tract were tested with this newly de- qPCR can be performed within two hours because of the signed qPCR assay and compared with culture results. elimination of postamplification handling. Of the samples, 132 (44.0%) were culture positive, In a recent study,10 qPCR was used to detect and whereas 151 (50.3%) tested positive using qPCR. The quantify U. parvum and U. urealyticum in specimens from U. parvum and U. urealyticum species were present in the lower genital tract. This test was evaluated in our 79.5% and 12.6% of the qPCR-positive samples, re- laboratory and did allow the differentiation and quantifi- spectively. Both species were found in 7.9% of those cation of U. urealyticum and U. parvum in two separate samples. Quantification of U. parvum and U. urea- reaction mixtures. However, cross-reactions with Staph- ؋ lyticum in the samples ranged from less than 2.5 ylococcus aureus DNA were encountered in the U. parvum ؋ 7 3 10 to 7.4 10 copies per specimen. In conclusion, mixture. the modified qPCR is a suitable method for rapid In this study, we investigated the nature of these cross- detection, differentiation, and quantification of U. reactions by sequence analysis of the nonspecific ampli- parvum and U. urealyticum. (J Mol Diagn 2011, 13: fication product, and we compared this sequence with 206–212; DOI: 10.1016/j.jmoldx.2010.10.007) the National Center for Biotechnology Information data- base. We designed new primers targeting the urease gene of U. parvum to improve the specificity of the qPCR. The genus Ureaplasma belongs to the Mycoplasmataceae Furthermore, the method was adapted to be more prac- family. Ureaplasmas are small prokaryotic cells that lack tical for a routine laboratory setting and the phocine her- a cell wall. Previously, there was only one known species found in humans (namely, Ureaplasma urealyticum) com- 1 prising 14 serotypes. In 2002, Robertson et al proposed Supported by a grant from Steunfonds Marguerite-Marie Delacroix. subdividing this species into U. parvum, comprising se- Accepted for publication October 13, 2010. rotypes 1, 3, 6, and 14; and U. urealyticum, comprising Address reprint requests to Ellen Vancutsem, MSc, Department of Micro- serotypes 2, 4, 5, and 7 through 13. These subtypes biology and Infection Control, Universitair Ziekenhuis Brussel, Laarbeeklaan cannot be distinguished from each other with routine 101, 1090 Brussels, Belgium. E-mail: [email protected]. 206 Quantitative PCR for Ureaplasma Species 207 JMD March 2011, Vol. 13, No. 2 pesvirus 1 (PhHV1) was included as an internal control to for primer specificity (http://www.ncbi.nlm.nih.gov, acces- monitor for the presence of inhibitory factors in the clinical sion date October 1, 2008). Primers were chosen within samples. the ureD subunit of the urease gene of U. parvum and U. urealyticum. One copy of this gene is present per cell. The amplicon product sizes were 147 and 146 bp for U. Materials and Methods parvum and U. urealyticum, respectively. Investigating the Issue of Specificity in the Original Method PCR Internal Control PCR inhibition and extraction were monitored by am- Reaction mixtures and cycling conditions were per- plifying the PhHV1. The use of this virus as a control for formed as previously described.10 Primer and probe se- extraction and the presence of amplification inhibitors quences were blasted with the GenBank database to were originally described by Niesters.11 The virus strain confirm specificity (http://www.ncbi.nlm.nih.gov, acces- was provided by the Department of Virology, University sion date April 25, 2008). Assay specificity was assessed Hospital Rotterdam, Rotterdam, the Netherlands. with the 14 reference strains of U. parvum (serotypes 1, 3, 6, and 14) and U. urealyticum (serotypes 2, 4, 5, and 7 through 13). In addition to the 14 reference strains of Modified qPCR Protocol Ureaplasma species, assay specificity was also deter- To detect both Ureaplasma species, real-time PCR was mined with 11 human Mycoplasma species, 29 bacte- performed in two separate 25-␮L reaction mixtures. The rial species, 1 yeast, and 1 viral pathogen: Acinetobac- PhHV1 viral DNA was detected in multiplex PCR with the ter baumannii, Bacteroides fragilis, Bordetella pertussis, two reaction mixtures for U. parvum and U. urealyticum Burkholderia cepacia, Citrobacter freundii, Chlamydia tracho- DNA. The reaction mixtures contained a ready-to-use 2x matis, Enterobacter aerogenes, Escherichia coli, Gardnerella real-time PCR reaction mix containing all necessary com- vaginalis, Herpes simplex virus 1/2, Listeria monocytogenes, ponents without primers and probes (IQ Multiplex Pow- Micrococcus species, Mobiluncus mulieris, Moraxella ca- ermix, Bio-Rad, Nazareth-Eke, Belgium), 0.35 ␮mol/L of tarrhalis, Mycoplasma hominis, Mycoplasma amphiforme, My- each primer, and 0.2 ␮mol/L of each probe (forward and coplasma fermentans, Mycoplasma genitalium, Mycoplasma reverse primer and probe of the target organism and of orale, Mycoplasma penetrans, Mycoplasma pirum, Myco- the internal control). DNA template, 2 ␮L, was added to plasma primatum, Mycoplasma salivarium, Mycoplasma the 23-␮L reaction mixtures. Probes (Taqman Minor spermatophilum, Mycoplasma pneumoniae, Pseudomonas Groove Binder) for U. parvum (5=-FAM-TTGACCACCCT- aeruginosa, Serratia marcescens, Stenotrophomonas mal- TACGAG-MGB-3=) and U. urealyticum (5=-FAM-TTGTC- tophilia, Streptococcus agalactiae (group B), Streptococ- CGCCTTTACGAG-MGB-3=) were manufactured by Ap- cus pneumoniae, Streptococcus pyogenes, Bordetella plied Biosystems (Warrington, UK). The Taqman probe parapertussis, Candida albicans, Hemophilus influenzae, for the internal control (Texas 5=-Red-TTTTTATGTGTC- Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus CGCCACCATCTGGATC-BHQ1-3=) was manufactured by species, Morganella morganii, Proteus mirabilis, Staphylo- Eurogentec (Luik, Belgium). Cycling conditions were as fol- coccus aureus oxacillin R, Staphylococcus aureus oxacillin lows: 95°C for 3 minutes, followed by 45 cycles of 95°C S, and Staphylococcus epidermidis. The last 11 organisms for 15 seconds and 58°C for 30 seconds. Amplification, are urease positive, which is important in specificity test- detection, and analysis were performed with a commer- ing because the urease gene is the target gene for cially available system (iCycler IQ real-time detection amplification. system; Bio-Rad). Data collection was performed during Nonspecific reactions were repeatedly tested (10-fold) the annealing step. The relative fluorescence measure- with reference strains (to confirm the presence of cross- ments were plotted against the cycle number for data reactions) and with two more clinical strains. Nonspecific analysis. Background fluorescence was corrected by amplification products were analyzed by cloning and se- manually setting the baseline cycles from 5 to 16. The quence analysis (ABI3730, Sanger sequencing, Bigdye log-linear portions of the amplification plots were used to v3.1; Applied Biosystems, Halle, Belgium) to retrieve the determine a fractional
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