# Rapid PCR Detection ofMycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum

Authors: Scott A. Cunningham; Jayawant N. Mandrekar; Jon E. Rosenblatt; Robin Patel
Journal: International Journal of Bacteriology
 (2013)

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## Abstract

Objective. We compared laboratory developed real-time PCR assays for detection of Mycoplasma hominis and for detection and differentiation of Ureaplasma urealyticum and parvum to culture using genitourinary specimens submitted for M. hominis and Ureaplasma culture. Methods. 283 genitourinary specimens received in the clinical bacteriology laboratory for M. hominis and Ureaplasma species culture were evaluated. Nucleic acids were extracted using the Total Nucleic Acid Kit on the MagNA Pure 2.0. 5 μL of the extracts were combined with 15 μL of each of the two master mixes. Assays were performed on the LightCycler 480 II system. Culture was performed using routine methods. Results.  M. hominis PCR detected 38/42 M. hominis culture-positive specimens, as well as 2 that were culture negative (sensitivity, 90.5%; specificity, 99.2%). Ureaplasma PCR detected 139/144 Ureaplasma culture-positive specimens, as well as 9 that were culture negative (sensitivity, 96.5%; specificity, 93.6%). Of the specimens that tested positive for Ureaplasma species, U. urealyticum alone was detected in 33, U. parvum alone in 109, and both in 6. Conclusion. The described PCR assays are rapid alternatives to culture for detection of M. hominis and Ureaplasma species, and, unlike culture, the Ureaplasma assay easily distinguishes U. urealyticum from parvum.


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## Body

## 1. Introduction

Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvumare small, fastidious bacteria belonging to the Mollicutes class. They lack a cell wall (preventing staining with Gram stain) and are not sensitively detected on routine bacterial cultures. Optimal recovery requires specialized media and growth conditions. There are several human pathogens in the genera Mycoplasmaand Ureaplasma which are responsible for a variety of clinical manifestations involving multiple body systems [1]. M. hominis causes septic arthritis and postpartum fever and has been associated with pelvic inflammatory disease and bacterial vaginosis [2]. Ureaplasmaspecies can cause acute urethritis and have been associated with bacterial vaginosis, preterm birth, and neonatal respiratory disease [1, 3].AlthoughM. hominis and Ureaplasmaspecies can be cultured, this requires technical skill for interpretation of microscopic colonies and takes two to five days. U. urealyticumwas the only Ureaplasma species until 2002, when U. parvum was described [4]. The two are not distinguished based on culture characteristics alone. Real-time PCR detection of these microorganisms from clinical samples circumvents technical issues related to culture and shortens turn-around time for detection and identification.Few real-time PCR assays and associated studies have been described forM. hominis.A real-time PCR assay targeting M. hominis gap identified two positive cervical swabs from women being evaluated for infertility [5]. 153 urogenital specimens were tested with a real-time PCR assay targeting M. hominis yidC, of which 45 were PCR- and culture positive and 10 PCR positive only [6]. Finally, extragenital M. hominisinfection was diagnosed in three patients using a real-time PCR assay targeting the M. hominis16S ribosomal RNA gene [7].There has been more work on real-time PCR assays forUreaplasmaspecies, although some have described assays but have not evaluated clinical specimens or clinical isolates [8]. A real-time PCR assay that detects and distinguishes U. urealyticumfrom parvumwas described but used to assess 87 vaginal swabs [9]. Tang et al. used a real-time PCR assay that detects and distinguishes U. parvumand urealyticum to test 346 genitourinary swabs; 120 were positive for the former and 21 for the latter, including 5 positive for both [10]. Finally, Vancutsem et al. used a real-time PCR assay for detection and differentiation of U. urealyticumand parvumto evaluate 300 lower genital tract specimens; 132 were culture positive, of which all plus an additional 19 were PCR-positive (19, U. urealyticum;120, U. parvum; 12, Ureaplasmaspecies) [11].Herein, we present one real-time PCR assay for the detection ofM. hominis and another for the detection and differentiation of Ureaplasmaspecies and report results of these assays on 283 genitourinary specimens in comparison to culture.


## 2. Materials and Methods

### 2.1. Clinical Specimens

283 genitourinary specimens (swabs, urine) submitted to the Mayo Clinic Clinical Microbiology Laboratory in transport medium (e.g., UTM, M5) forM. hominis and Ureaplasmaculture were evaluated. No clinical data associated with these specimens was available. This study was approved by the Mayo Clinic Institutional Review Board.


### 2.2.Mycoplasma hominis Culture

Samples were placed into arginine broth, incubated at 35°C, and monitored four times daily for up to five days. Color change (indicating an alkaline pH shift) in the arginine broth prompted subculture of 50μL to an A7 agar plate. Plates were incubated anaerobically at 35°C for up to five days and examined daily with an inverted light microscope for “fried egg” morphology colonies.


### 2.3.Ureaplasma Culture

Samples were placed into U9 broth, incubated at 35°C, and monitored four times daily for up to five days. A color change (indicating an alkaline pH shift) in the U9 broth prompted subculture of 100μL to an A7 agar plate. Plates were incubated anaerobically at 35°C for up to 48 hours and examined with an inverted light microscope for small, circular to irregular colonies growing into the surface of the agar, with a surrounding red zone. Confirmation of Ureaplasmaspecies was indicated by golden-brown stained colonies with the addition of 0.167 M CO(NH2)2 and 0.04 M MnCl2 in water.


### 2.4. Sample Processing for PCR

Samples were vortexed and 200μL transferred to a MagNA Pure sample cartridge (Roche Applied Science, Indianapolis, IN). DNA extraction was performed on the MagNA Pure LC 2.0 using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science) with a final elution volume of 100 μL.


### 2.5. Polymerase Chain Reaction Assay

Primers and probes (Table1) were designed using the LightCycler Probe Design Software, version 2.0 (Roche Diagnostics, Indianapolis, IN, USA) and DNA Workbench, version 5.7.1 (CLC Bio, Cambridge, MA, USA). Positive control plasmids were constructed for the three target-specific genes (Table 1) using the pCR 2.1 TOPO TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA, USA). Sources for the inserted target sequences were M. hominisATCC 23114, U. urealyticumATCC 27618, and U. parvumATCC 27815D. Plasmids were purified using the High Pure Plasmid Isolation Kit (Roche Applied Science). Sizes of the cloned inserts were confirmed by EcoR1 digestion. Plasmid inserts were sequenced using M13 forward and reverse primers included in the cloning kit, to confirm proper insert orientation. Plasmids were diluted in Tris-EDTA buffer (pH 8.0) and stored at 4°C.Table 1
Primers and probes.
Mycoplasma hominis
tuf (set number 793, TIB MolBio, Aldelphia, NJ; 10X concentration)a
Primers
tuf 193F
5′ AATTGATATGTTTAAAGATGATGAAAGAGA 3′
tuf 193R
5′ TGTATCAACAGCATCCATTAATTCC 3′
Probes
tuf 193flb
5′ GACGTAAGAAGCCTTCTATCAGAATATGGT FITC 3′
tuf 193iLC610c
5′ Red610 TGATGGTGACAATGCTCCTATTATTGCTGGTTC PO4 3′
Ureaplasmaspecies ureC (set number 684, TIB MolBio; 10X concentration)d
ureC 158F
5′ CCTGCTTCGTTTAATGTATCTG 3′
Primers
ureC 158R
5′ GAAGATCCAATCTTTGAACAAATCGTA 3′
ureC 158R5
5′ GAAGATCCAATCTTTGAACAAATTGCT 3′
Probes
ureC 158flb
5′ AGCAACTGTTAATGCTAAGTCAATAGCGTTTCCTG FITC 3′
ureC 158iLC610c
5′ Red610 GCCCCTCAGTCTTCGTGAATCTTAAGACCACAAGC PO4 3′
atuf target corresponds to 66720–66912 of GenBank accession number FP236530.
bLabeled with fluorescein on 3′ end.
cLabeled with LC610 on 5′ end and a phosphate on 3′ end.
d
ureC target corresponds to 527786–527943 of GenBank accession number CP001184.The two assays were independently optimized on the LightCycler 480 II platform employing LightCycler 480 Software version 1.5 (Roche Applied Science). 15μL of PCR master mix, containing final concentrations of 1X Roche Genotyping Master (Taq DNA polymerase, PCR reaction buffer, deoxyribonucleoside triphosphate with dUTP substituted for dTTP and 1 mM MgCl2), 1 mM (additional) MgCl2, and 1X of each of the LightCycler primer-probe sets (Table 1) were added to a 96-well LightCycler 480 plate. Extracted nucleic acid (5 μL) was then added to each well. The cycling program was as follows: denaturation at 95°C for 10 min; amplification for 45 cycles of 10 s at 95°C, 15 s at 55°C (single acquisition), and 15 s at 72°C; melting curve analysis for 30 s at 95°C, 10 s at 59°C, 15 s at 45°C (ramp rate of 0.1°C/s), and 0 s at 80°C (ramp rate of 0.14°C/s and continuous acquisition); and cooling for 30 s at 40°C. Positive and negative controls were included in each run. The positive control consisted of the abovementioned plasmids in S.T.A.R. buffer : sterile water (1 : 1) at a concentration of 1,000 targets/μL. The negative control consisted of 1,000 colony forming units of Escherichia coli ATCC 25922 S.T.A.R. buffer : sterile water (1 : 1) at a concentration of 1,000 targets/μL.


### 2.6. Polymerase Chain Reaction Sensitivity and Specificity

Predicted amplified product, primer, and probe sequences were subjected to BLAST searches using the National Center for Biotechnology Information (NCBI) genomic database (http://www.ncbi.nlm.nih.gov/). Analytical sensitivity was assessed by spiking a series of six tenfold dilutions of quantified genomic DNA from M. hominis ATCC 23114, U. urealyticum ATCC 27816, and U. parvum ATCC 27815D into genitourinary samples. Each dilution was extracted in triplicate and each extract was assayed in duplicate. The limit of detection was the lowest dilution where all six replicates were detected. Inclusivity and cross-reactivity were assessed using a panel organisms (Table 2), including 16 members of the Mollicutes class.Table 2
Cross-reactivity and inclusivity panel. OnlyMycoplasma hominis, Ureaplasma parvum,and Ureaplasma urealyticumwere detected (by the appropriate assays).
Organism
Accession no. or source
Organism
Accession no. or source
Acholeplasma laidlawii
ATCC 23206
Entamoeba histolytica
ATCC 30459
Acinetobacter baumannii
ATCC 19606
Entamoeba moshkovskii
ATCC 30042
Acinetobacter lwoffii/haemolyticus
QC Strain
Enterobacter cloacae
ATCC 13047
Actinomyces odontolyticus
ATCC 17929
Enterococcus faecalis
ATCC19433U
Aeromonas hydrophila
CAP-D-1-82
Enterococcus faecium
ATCC 19434
Arcanobacterium haemolyticum
ATCC 9345
Escherichia coli
ATCC 25922
Arcanobacterium pyogenes
ATCC 19411
Escherichia coli O142:K86(B):H6
ATCC 23985
Parabacteroides distasonis
ATCC 8503
Escherichia coliO157:H7
ATCC 35150
Bacteroides fragilis
ATCC 25285
Escherichia coliO70:K:H42
ATCC 23533
Bacteroides thetaiotaomicron
ATCC 29741
Escherichia fergusonii
ATCC 35469
Bacteroides vulgatus
ATCC 29327
Escherichia hermannii
ATCC 33650
Bifidobacterium adolescentis
ATCC 15703
Escherichia vulneris
ATCC 33821
Bifidobacterium bifidum
ATCC 29521
Eubacterium rectale
ATCC 33656
Bordetella bronchiseptica
ATCC 19395
Finegoldia magna
ATCC 29328
Bordetella holmesii
ATCC 51541
Fluoribacter bozemanae
ATCC 33217
Bordetella parapertussis
ATCC 15311
Fluoribacter gormanii
ATCC 33297
Bordetella pertussis
ATCC 9797
Fusobacterium nucleatum
ATCC 25559
Burkholderia cepacia
SCB1277
Gardnerella vaginalis
NYS 4-87
Campylobacter coli
ATCC 33559
Giardia lamblia
ATCC 30957
Campylobacter jejuni
ATCC 33560
Haemophilus influenzae
ATCC 10211
Chlamydia trachomatis
ATCC VR-348B
Human DNA
MRC-5 cells
Chlamydophila pneumoniae
ATCC 53592
Klebsiella oxytoca
ATCC 700324
Chlamydophila pneumoniae
ATCC VR-1310
Klebsiella pneumoniae
ATCC 700603
Citrobacter freundii
ATCC 8090
Lactobacillus delbrueckiissp.
lactis
ATCC 12315
Clostridium difficile
ATCC 9689
Lactobacillus rhamnosus
ATCC 7469
Clostridium perfringens
ATCC 13124
Fluoribacter dumoffii
ATCC 33279
Clostridium ramosum
ATCC 25582
Legionella jordanis
ATCC 33623
Collinsella aerofaciens
ATCC 25986
Legionella longbeachae
ATCC 33462
Corynebacterium diphtheriae
SCB-25-86
Tatlockia micdadei
ATCC 33204
Corynebacterium pseudodiphtheria
NY-4-88
Legionella pneumophila
ATCC 33152
Cryptosporidiumspecies
feline isolate
Legionella wadsworthii
ATCC 33877
Dientamoeba fragilis
ATCC 30948
Listeria monocytogenes
ATCC 15313
Eggerthella lenta
ATCC 25559
Moraxella catarrhalis
ATCC 8176
Encephalitozoon cuniculi
JS strain
Morganella morganii
CAP-D-5-79
Encephalitozoon hellem
ATCC 50451
Mycobacterium africanum
ATCC 25420
Encephalitozoon intestinalis
ATCC 50651
Mycobacterium avium
ATCC 700398
Mycobacterium avium
ATCC 700897
Proteus mirabilis
ATCC 35659
Mycobacterium bovis
ATCC 19210
Proteus vulgaris
QC strain
Mycobacterium bovis(BCG)
ATCC 35735
Pseudomonas aeruginosa
ATCC 27853
Mycobacterium gordonae
ATCC 14470
Pseudomonas fluorescens/putida
CDC-AB4-B10-84
Mycobacterium intracellulare
ATCC 35761
Rhodococcus equi
ATCC 6939
Mycobacterium kansasii
ATCC 12478
Salmonella enterica
ATCC 35987
Mycobacterium microti
ATCC 19422
Salmonella serogroup B
CAP-D-1-69
Mycobacterium smegmatis
ATCC 19980
Shigella dysenteriae
CDC 82-002-72
Mycobacterium tuberculosis
ATCC 25177
Shigella flexneriserotype 2a
ATCC29903
Mycobacterium tuberculosis
ATCC 27294
Shigella sonnei
ATCC 25931
Mycobacterium tuberculosis
ATCC 35825
Staphylococcus aureus
ATCC 25923
Mycobacterium tuberculosis
ATCC 35837
Staphylococcus epidermidis
ATCC 14990
Mycoplasma arginini
ATCC 23838D
Stenotrophomonas maltophilia
SCB-33-77
Mycoplasma arthritidis
ATCC 19611D
Streptococcus bovis
CAP-D-16-83
Mycoplasma bovis
ATCC 25523D
Streptococcus pneumoniae
ATCC 49619
Mycoplasma buccale
ATCC 23636
Streptococcus pyogenes
ATCC 19615
Mycoplasma faucium
ATCC 25293
Streptococcus sanguinis
ATCC 10556
Mycoplasma fermentans
ATCC 19989
*Ureaplasma parvum
ATCC 28715
Mycoplasma genitalium
ATTC 33530
*Ureaplasma urealyticum
ATCC 27618
*Mycoplasma hominis
ATCC 23114
Yersinia enterocolitica
ATCC 9610
Mycoplasma hyorhinis
ATCC 17981D
BK polyomavirus
ATCC VR-837
Mycoplasma lipophilum
ATCC 27104
Cytomegalovirus
ATCC VR-538
Mycoplasma orale
ATCC 23714
Mycoplasma phocidae
ATCC 33657
Herpes simplex virus 1
Lab Control
Mycoplasma pirum
ATCC 25960D
Herpes simplex virus 2
Lab Control
Mycoplasma pneumoniae
ATCC 15531D
Human adenovirus 9
ATCC VR-1086
Mycoplasma salivarium
ATCC 23064
Human coronavirus 229E
ATCC VR-740
Neisseria gonorrhoeae
ATCC 43069
Human coxsackievirus B 1 (Enterovirus)
ATCC VR-28
Neisseria lactamica
ATCC 23970
Human herpesvirus 6B
ATCC VR-1467
Neisseria meningitidis
ATCC 13077
Human herpesvirus 7
ABI 08765000
Nocardia brasiliensis
ATCC 51512
Human herpesvirus 8
ABI 08735000
Nocardia brevicatena
ATCC 15333
Human parainfluenza virus 1
ATCC VR-94
Nocardia carnea
ATCC 6847
Human parainfluenza virus 3
ATCC VR-93
Nocardiopsis dassonvillei
ATCC 23218
Respiratory syncytial virus A2
ATCC VR-1540
Nocardia farcinica
ATCC 3318
Respiratory syncytial virus B
ATCC VR-1401
Nocardia otitidiscaviarum
ATCC 14629
Influenza A virus (H3N2)
ATCC VR-810
Nocardia transvalensis
ATCC 6865
Influenza B virus
ATCC VR-791
Plesiomonas shigelloides
ATCC 14029
Measles virus
ATCC VR-24
Porphyromonas gingivalis
ATCC 33277
Mumps virus
ATCC VR-365
Prevotella melaninogenica
ATCC 25845
Varicella-zoster virus
ATCC VR-1367
Prevotella oralis
ATCC 33269Clinical sensitivity and specificity were assessed by assaying the aforementioned clinical specimens and comparing results to those of culture. Discordant samples were tested courtesy of Dr. Stellrecht, at an independent clinical laboratory (Albany Medical Center) with a previously described assay [12].The ability of theUreaplasmaassay to differentiate urealyticumfrom parvumwas assessed as follows. Cultured isolates from clinical samples were directly subjected to PCR with species differentiation based on melting curve analysis; sequence variations underlying the probed regions of U. urealyticum andparvum result in separation of the melting temperature of the two species (Figures 1 and 2). Results were compared to those of a previously described conventional PCR speciation method targeting the multiple-banded antigen using primers UMS-57 and UMA222 for U. parvum and UMS-170 and UMA263 for U. urealyticum [13].Figure 1
Melting curves ofUreaplasma urealyticum and Ureaplasma parvum.Figure 2
Ureaplasmaprobe design.


### 2.7. Statistical Analysis

Assessment of the assays’ sensitivity and specificity, with associated 95% confidence intervals (CI), compared to that of culture forM. hominisand Ureaplasmaspecies was made using SAS software version 9.1 (SAS, INC, Cary, NC, USA).


## 2.1. Clinical Specimens

283 genitourinary specimens (swabs, urine) submitted to the Mayo Clinic Clinical Microbiology Laboratory in transport medium (e.g., UTM, M5) forM. hominis and Ureaplasmaculture were evaluated. No clinical data associated with these specimens was available. This study was approved by the Mayo Clinic Institutional Review Board.


## 2.2.Mycoplasma hominis Culture

Samples were placed into arginine broth, incubated at 35°C, and monitored four times daily for up to five days. Color change (indicating an alkaline pH shift) in the arginine broth prompted subculture of 50μL to an A7 agar plate. Plates were incubated anaerobically at 35°C for up to five days and examined daily with an inverted light microscope for “fried egg” morphology colonies.


## 2.3.Ureaplasma Culture

Samples were placed into U9 broth, incubated at 35°C, and monitored four times daily for up to five days. A color change (indicating an alkaline pH shift) in the U9 broth prompted subculture of 100μL to an A7 agar plate. Plates were incubated anaerobically at 35°C for up to 48 hours and examined with an inverted light microscope for small, circular to irregular colonies growing into the surface of the agar, with a surrounding red zone. Confirmation of Ureaplasmaspecies was indicated by golden-brown stained colonies with the addition of 0.167 M CO(NH2)2 and 0.04 M MnCl2 in water.


## 2.4. Sample Processing for PCR

Samples were vortexed and 200μL transferred to a MagNA Pure sample cartridge (Roche Applied Science, Indianapolis, IN). DNA extraction was performed on the MagNA Pure LC 2.0 using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science) with a final elution volume of 100 μL.


## 2.5. Polymerase Chain Reaction Assay

Primers and probes (Table1) were designed using the LightCycler Probe Design Software, version 2.0 (Roche Diagnostics, Indianapolis, IN, USA) and DNA Workbench, version 5.7.1 (CLC Bio, Cambridge, MA, USA). Positive control plasmids were constructed for the three target-specific genes (Table 1) using the pCR 2.1 TOPO TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA, USA). Sources for the inserted target sequences were M. hominisATCC 23114, U. urealyticumATCC 27618, and U. parvumATCC 27815D. Plasmids were purified using the High Pure Plasmid Isolation Kit (Roche Applied Science). Sizes of the cloned inserts were confirmed by EcoR1 digestion. Plasmid inserts were sequenced using M13 forward and reverse primers included in the cloning kit, to confirm proper insert orientation. Plasmids were diluted in Tris-EDTA buffer (pH 8.0) and stored at 4°C.Table 1
Primers and probes.
Mycoplasma hominis
tuf (set number 793, TIB MolBio, Aldelphia, NJ; 10X concentration)a
Primers
tuf 193F
5′ AATTGATATGTTTAAAGATGATGAAAGAGA 3′
tuf 193R
5′ TGTATCAACAGCATCCATTAATTCC 3′
Probes
tuf 193flb
5′ GACGTAAGAAGCCTTCTATCAGAATATGGT FITC 3′
tuf 193iLC610c
5′ Red610 TGATGGTGACAATGCTCCTATTATTGCTGGTTC PO4 3′
Ureaplasmaspecies ureC (set number 684, TIB MolBio; 10X concentration)d
ureC 158F
5′ CCTGCTTCGTTTAATGTATCTG 3′
Primers
ureC 158R
5′ GAAGATCCAATCTTTGAACAAATCGTA 3′
ureC 158R5
5′ GAAGATCCAATCTTTGAACAAATTGCT 3′
Probes
ureC 158flb
5′ AGCAACTGTTAATGCTAAGTCAATAGCGTTTCCTG FITC 3′
ureC 158iLC610c
5′ Red610 GCCCCTCAGTCTTCGTGAATCTTAAGACCACAAGC PO4 3′
atuf target corresponds to 66720–66912 of GenBank accession number FP236530.
bLabeled with fluorescein on 3′ end.
cLabeled with LC610 on 5′ end and a phosphate on 3′ end.
d
ureC target corresponds to 527786–527943 of GenBank accession number CP001184.The two assays were independently optimized on the LightCycler 480 II platform employing LightCycler 480 Software version 1.5 (Roche Applied Science). 15μL of PCR master mix, containing final concentrations of 1X Roche Genotyping Master (Taq DNA polymerase, PCR reaction buffer, deoxyribonucleoside triphosphate with dUTP substituted for dTTP and 1 mM MgCl2), 1 mM (additional) MgCl2, and 1X of each of the LightCycler primer-probe sets (Table 1) were added to a 96-well LightCycler 480 plate. Extracted nucleic acid (5 μL) was then added to each well. The cycling program was as follows: denaturation at 95°C for 10 min; amplification for 45 cycles of 10 s at 95°C, 15 s at 55°C (single acquisition), and 15 s at 72°C; melting curve analysis for 30 s at 95°C, 10 s at 59°C, 15 s at 45°C (ramp rate of 0.1°C/s), and 0 s at 80°C (ramp rate of 0.14°C/s and continuous acquisition); and cooling for 30 s at 40°C. Positive and negative controls were included in each run. The positive control consisted of the abovementioned plasmids in S.T.A.R. buffer : sterile water (1 : 1) at a concentration of 1,000 targets/μL. The negative control consisted of 1,000 colony forming units of Escherichia coli ATCC 25922 S.T.A.R. buffer : sterile water (1 : 1) at a concentration of 1,000 targets/μL.


## 2.6. Polymerase Chain Reaction Sensitivity and Specificity

Predicted amplified product, primer, and probe sequences were subjected to BLAST searches using the National Center for Biotechnology Information (NCBI) genomic database (http://www.ncbi.nlm.nih.gov/). Analytical sensitivity was assessed by spiking a series of six tenfold dilutions of quantified genomic DNA from M. hominis ATCC 23114, U. urealyticum ATCC 27816, and U. parvum ATCC 27815D into genitourinary samples. Each dilution was extracted in triplicate and each extract was assayed in duplicate. The limit of detection was the lowest dilution where all six replicates were detected. Inclusivity and cross-reactivity were assessed using a panel organisms (Table 2), including 16 members of the Mollicutes class.Table 2
Cross-reactivity and inclusivity panel. OnlyMycoplasma hominis, Ureaplasma parvum,and Ureaplasma urealyticumwere detected (by the appropriate assays).
Organism
Accession no. or source
Organism
Accession no. or source
Acholeplasma laidlawii
ATCC 23206
Entamoeba histolytica
ATCC 30459
Acinetobacter baumannii
ATCC 19606
Entamoeba moshkovskii
ATCC 30042
Acinetobacter lwoffii/haemolyticus
QC Strain
Enterobacter cloacae
ATCC 13047
Actinomyces odontolyticus
ATCC 17929
Enterococcus faecalis
ATCC19433U
Aeromonas hydrophila
CAP-D-1-82
Enterococcus faecium
ATCC 19434
Arcanobacterium haemolyticum
ATCC 9345
Escherichia coli
ATCC 25922
Arcanobacterium pyogenes
ATCC 19411
Escherichia coli O142:K86(B):H6
ATCC 23985
Parabacteroides distasonis
ATCC 8503
Escherichia coliO157:H7
ATCC 35150
Bacteroides fragilis
ATCC 25285
Escherichia coliO70:K:H42
ATCC 23533
Bacteroides thetaiotaomicron
ATCC 29741
Escherichia fergusonii
ATCC 35469
Bacteroides vulgatus
ATCC 29327
Escherichia hermannii
ATCC 33650
Bifidobacterium adolescentis
ATCC 15703
Escherichia vulneris
ATCC 33821
Bifidobacterium bifidum
ATCC 29521
Eubacterium rectale
ATCC 33656
Bordetella bronchiseptica
ATCC 19395
Finegoldia magna
ATCC 29328
Bordetella holmesii
ATCC 51541
Fluoribacter bozemanae
ATCC 33217
Bordetella parapertussis
ATCC 15311
Fluoribacter gormanii
ATCC 33297
Bordetella pertussis
ATCC 9797
Fusobacterium nucleatum
ATCC 25559
Burkholderia cepacia
SCB1277
Gardnerella vaginalis
NYS 4-87
Campylobacter coli
ATCC 33559
Giardia lamblia
ATCC 30957
Campylobacter jejuni
ATCC 33560
Haemophilus influenzae
ATCC 10211
Chlamydia trachomatis
ATCC VR-348B
Human DNA
MRC-5 cells
Chlamydophila pneumoniae
ATCC 53592
Klebsiella oxytoca
ATCC 700324
Chlamydophila pneumoniae
ATCC VR-1310
Klebsiella pneumoniae
ATCC 700603
Citrobacter freundii
ATCC 8090
Lactobacillus delbrueckiissp.
lactis
ATCC 12315
Clostridium difficile
ATCC 9689
Lactobacillus rhamnosus
ATCC 7469
Clostridium perfringens
ATCC 13124
Fluoribacter dumoffii
ATCC 33279
Clostridium ramosum
ATCC 25582
Legionella jordanis
ATCC 33623
Collinsella aerofaciens
ATCC 25986
Legionella longbeachae
ATCC 33462
Corynebacterium diphtheriae
SCB-25-86
Tatlockia micdadei
ATCC 33204
Corynebacterium pseudodiphtheria
NY-4-88
Legionella pneumophila
ATCC 33152
Cryptosporidiumspecies
feline isolate
Legionella wadsworthii
ATCC 33877
Dientamoeba fragilis
ATCC 30948
Listeria monocytogenes
ATCC 15313
Eggerthella lenta
ATCC 25559
Moraxella catarrhalis
ATCC 8176
Encephalitozoon cuniculi
JS strain
Morganella morganii
CAP-D-5-79
Encephalitozoon hellem
ATCC 50451
Mycobacterium africanum
ATCC 25420
Encephalitozoon intestinalis
ATCC 50651
Mycobacterium avium
ATCC 700398
Mycobacterium avium
ATCC 700897
Proteus mirabilis
ATCC 35659
Mycobacterium bovis
ATCC 19210
Proteus vulgaris
QC strain
Mycobacterium bovis(BCG)
ATCC 35735
Pseudomonas aeruginosa
ATCC 27853
Mycobacterium gordonae
ATCC 14470
Pseudomonas fluorescens/putida
CDC-AB4-B10-84
Mycobacterium intracellulare
ATCC 35761
Rhodococcus equi
ATCC 6939
Mycobacterium kansasii
ATCC 12478
Salmonella enterica
ATCC 35987
Mycobacterium microti
ATCC 19422
Salmonella serogroup B
CAP-D-1-69
Mycobacterium smegmatis
ATCC 19980
Shigella dysenteriae
CDC 82-002-72
Mycobacterium tuberculosis
ATCC 25177
Shigella flexneriserotype 2a
ATCC29903
Mycobacterium tuberculosis
ATCC 27294
Shigella sonnei
ATCC 25931
Mycobacterium tuberculosis
ATCC 35825
Staphylococcus aureus
ATCC 25923
Mycobacterium tuberculosis
ATCC 35837
Staphylococcus epidermidis
ATCC 14990
Mycoplasma arginini
ATCC 23838D
Stenotrophomonas maltophilia
SCB-33-77
Mycoplasma arthritidis
ATCC 19611D
Streptococcus bovis
CAP-D-16-83
Mycoplasma bovis
ATCC 25523D
Streptococcus pneumoniae
ATCC 49619
Mycoplasma buccale
ATCC 23636
Streptococcus pyogenes
ATCC 19615
Mycoplasma faucium
ATCC 25293
Streptococcus sanguinis
ATCC 10556
Mycoplasma fermentans
ATCC 19989
*Ureaplasma parvum
ATCC 28715
Mycoplasma genitalium
ATTC 33530
*Ureaplasma urealyticum
ATCC 27618
*Mycoplasma hominis
ATCC 23114
Yersinia enterocolitica
ATCC 9610
Mycoplasma hyorhinis
ATCC 17981D
BK polyomavirus
ATCC VR-837
Mycoplasma lipophilum
ATCC 27104
Cytomegalovirus
ATCC VR-538
Mycoplasma orale
ATCC 23714
Mycoplasma phocidae
ATCC 33657
Herpes simplex virus 1
Lab Control
Mycoplasma pirum
ATCC 25960D
Herpes simplex virus 2
Lab Control
Mycoplasma pneumoniae
ATCC 15531D
Human adenovirus 9
ATCC VR-1086
Mycoplasma salivarium
ATCC 23064
Human coronavirus 229E
ATCC VR-740
Neisseria gonorrhoeae
ATCC 43069
Human coxsackievirus B 1 (Enterovirus)
ATCC VR-28
Neisseria lactamica
ATCC 23970
Human herpesvirus 6B
ATCC VR-1467
Neisseria meningitidis
ATCC 13077
Human herpesvirus 7
ABI 08765000
Nocardia brasiliensis
ATCC 51512
Human herpesvirus 8
ABI 08735000
Nocardia brevicatena
ATCC 15333
Human parainfluenza virus 1
ATCC VR-94
Nocardia carnea
ATCC 6847
Human parainfluenza virus 3
ATCC VR-93
Nocardiopsis dassonvillei
ATCC 23218
Respiratory syncytial virus A2
ATCC VR-1540
Nocardia farcinica
ATCC 3318
Respiratory syncytial virus B
ATCC VR-1401
Nocardia otitidiscaviarum
ATCC 14629
Influenza A virus (H3N2)
ATCC VR-810
Nocardia transvalensis
ATCC 6865
Influenza B virus
ATCC VR-791
Plesiomonas shigelloides
ATCC 14029
Measles virus
ATCC VR-24
Porphyromonas gingivalis
ATCC 33277
Mumps virus
ATCC VR-365
Prevotella melaninogenica
ATCC 25845
Varicella-zoster virus
ATCC VR-1367
Prevotella oralis
ATCC 33269Clinical sensitivity and specificity were assessed by assaying the aforementioned clinical specimens and comparing results to those of culture. Discordant samples were tested courtesy of Dr. Stellrecht, at an independent clinical laboratory (Albany Medical Center) with a previously described assay [12].The ability of theUreaplasmaassay to differentiate urealyticumfrom parvumwas assessed as follows. Cultured isolates from clinical samples were directly subjected to PCR with species differentiation based on melting curve analysis; sequence variations underlying the probed regions of U. urealyticum andparvum result in separation of the melting temperature of the two species (Figures 1 and 2). Results were compared to those of a previously described conventional PCR speciation method targeting the multiple-banded antigen using primers UMS-57 and UMA222 for U. parvum and UMS-170 and UMA263 for U. urealyticum [13].Figure 1
Melting curves ofUreaplasma urealyticum and Ureaplasma parvum.Figure 2
Ureaplasmaprobe design.


## 2.7. Statistical Analysis

Assessment of the assays’ sensitivity and specificity, with associated 95% confidence intervals (CI), compared to that of culture forM. hominisand Ureaplasmaspecies was made using SAS software version 9.1 (SAS, INC, Cary, NC, USA).


## 3. Results

### 3.1. Polymerase Chain Reaction Sensitivity and Specificity

The analytical sensitivity of both assays was 100 genome copies/μL genitourinary specimen. Amplified product, primer, and probe sequences were subjected to NCBI database searches using BLAST software; no significant homology was noted outside of the genera targeted by these assays. Nucleic acid material from members of the Mollicutes class, excluding M. hominis and the Ureaplasmaspecies, was not detected (Table 2).


### 3.2. Clinical Sensitivity and Specificity

TheM. hominisPCR assay had a clinical sensitivity and specificity of 90.7% (95% CI: 77.4%, 97.3%) and 99.2% (95% CI: 97.0%, 99.9%), respectively (Table 3). The 6 discordant results were tested at the Albany Medical Center using an assay targeting the 16S ribosomal RNA gene; [12] both PCR positive/culture-negative specimens were PCR positive, and three of four PCR negative/culture-positive specimens were PCR negative.Table 3
Comparison of PCR with culture forMycoplasma hominisand Ureaplasmaspecies detection.
M. hominis culture
Positive
Negative
M. hominisPCR
Positive
38
2
40
Negative
4
239
243
42
241
283
Sensitivity = 90.5% (95% CI:  77.4%, 97.3%), specificity = 99.2% (95% CI:  97.0%, 99.9%)
Ureaplasma species culture
Positive
Negative
UreaplasmaPCR
Positive
1391
92
148
Negative
5
130
135
144
139
283
Sensitivity = 96.5% (95% CI:  92.1%, 98.9%), specificity = 93.5% (95% CI:  88.1%, 97.0%)
1U. urealyticum (n=28), U. parvum (n=109), U. urealyticum and U. parvum (2).
2
U. urealyticum (n=3), U. parvum (n=6).TheUreaplasmaPCR assay had a clinical sensitivity and specificity of 96.5% (95% CI: 92.1%, 98.9%) and 93.8% (95% CI: 88.1%, 97.0%), respectively (Table 3). The 14 discordant results were tested at Albany Medical Center; [12] five of nine specimens that were PCR positive/culture negative were PCR positive, and all five specimens that were PCR negative/culture positive were PCR negative. Of the specimens that tested positive for Ureaplasmaspecies by PCR and were culture positive, U. urealyticumalone was detected in 28, U. parvum alone in 109, and both in 2. Among the PCR positive/culture-negative specimens, U. urealyticum was detected in 3 and U. parvum in 6.Thirty-one culture isolates ofUreaplasmaspecies were tested with the Ureaplasma assay and a previously reported PCR method that differentiates between the two species [13]. The reference method yielded species-level identification for 20 isolates, including 4 U. urealyticumand 16 U. parvum, with identical results to the assay described herein.The remaining 11 isolates were speciated by the assay described herein but not by the reference method; they were confirmed to be Ureaplasmaspecies by partial 16S ribosomal RNA gene sequencing [14]. All partial 16S ribosomal RNA gene sequences were identical to one another and were perfect matches to bases 145,365 through 145,845 of GenBank AF222894.1 (U. parvum) and bases 40 through 520 of GenBank L08642.1 (U. urealyticum).


## 3.1. Polymerase Chain Reaction Sensitivity and Specificity

The analytical sensitivity of both assays was 100 genome copies/μL genitourinary specimen. Amplified product, primer, and probe sequences were subjected to NCBI database searches using BLAST software; no significant homology was noted outside of the genera targeted by these assays. Nucleic acid material from members of the Mollicutes class, excluding M. hominis and the Ureaplasmaspecies, was not detected (Table 2).


## 3.2. Clinical Sensitivity and Specificity

TheM. hominisPCR assay had a clinical sensitivity and specificity of 90.7% (95% CI: 77.4%, 97.3%) and 99.2% (95% CI: 97.0%, 99.9%), respectively (Table 3). The 6 discordant results were tested at the Albany Medical Center using an assay targeting the 16S ribosomal RNA gene; [12] both PCR positive/culture-negative specimens were PCR positive, and three of four PCR negative/culture-positive specimens were PCR negative.Table 3
Comparison of PCR with culture forMycoplasma hominisand Ureaplasmaspecies detection.
M. hominis culture
Positive
Negative
M. hominisPCR
Positive
38
2
40
Negative
4
239
243
42
241
283
Sensitivity = 90.5% (95% CI:  77.4%, 97.3%), specificity = 99.2% (95% CI:  97.0%, 99.9%)
Ureaplasma species culture
Positive
Negative
UreaplasmaPCR
Positive
1391
92
148
Negative
5
130
135
144
139
283
Sensitivity = 96.5% (95% CI:  92.1%, 98.9%), specificity = 93.5% (95% CI:  88.1%, 97.0%)
1U. urealyticum (n=28), U. parvum (n=109), U. urealyticum and U. parvum (2).
2
U. urealyticum (n=3), U. parvum (n=6).TheUreaplasmaPCR assay had a clinical sensitivity and specificity of 96.5% (95% CI: 92.1%, 98.9%) and 93.8% (95% CI: 88.1%, 97.0%), respectively (Table 3). The 14 discordant results were tested at Albany Medical Center; [12] five of nine specimens that were PCR positive/culture negative were PCR positive, and all five specimens that were PCR negative/culture positive were PCR negative. Of the specimens that tested positive for Ureaplasmaspecies by PCR and were culture positive, U. urealyticumalone was detected in 28, U. parvum alone in 109, and both in 2. Among the PCR positive/culture-negative specimens, U. urealyticum was detected in 3 and U. parvum in 6.Thirty-one culture isolates ofUreaplasmaspecies were tested with the Ureaplasma assay and a previously reported PCR method that differentiates between the two species [13]. The reference method yielded species-level identification for 20 isolates, including 4 U. urealyticumand 16 U. parvum, with identical results to the assay described herein.The remaining 11 isolates were speciated by the assay described herein but not by the reference method; they were confirmed to be Ureaplasmaspecies by partial 16S ribosomal RNA gene sequencing [14]. All partial 16S ribosomal RNA gene sequences were identical to one another and were perfect matches to bases 145,365 through 145,845 of GenBank AF222894.1 (U. parvum) and bases 40 through 520 of GenBank L08642.1 (U. urealyticum).


## 4. Discussion

We describe two rapid real-time PCR assays, one for detection ofM. hominisand the other for detection of Ureaplasmaspecies; they have comparable performance to culture but yield results in three hours, instead of two to five days for culture. These assays are performed on a standard platform and are adaptable to automation, a potential advantage over other described methods, especially for large reference laboratories that process large numbers of specimens.We are not aware of other real-time PCR studies that have assessedM. hominisand Ureaplasmaspecies using the same set of clinical samples. Overall, 14% of tested specimens were PCR positive for M. hominisand 52% for Ureaplasmaspecies. A multiplex PCR enzyme-linked immunosorbent assay was used to detect M. hominis and U. parvum and urealyticum in cervical swabs from 175 Australian women with and without cervicitis; 16% tested positive for M. hominis and 68% for Ureaplasmaspecies [15]. Multiplex PCR and autocapillary electrophoresis were used to detect M. hominisand Ureaplasmaspecies (without differentiating U. parvumfrom urealyticum) in genitourinary specimens from 113 South Koreans with sexually transmitted infections; 12% were positive for M. hominis and 43% for Ureaplasmaspecies [16]. These findings are similar to ours [15, 16].Our PCR assay not only detectsUreaplasmaspecies but also differentiates U. parvumfrom urealyticum.As in prior studies, U. parvumwas more common than U. urealyticum, [10, 11, 15, 17] with 41% of the genitourinary specimens testing positive for the former and 12% for the latter.In one prior study, 63% of specimens were positive for U. parvumand 7% for U. urealyticum [15]. Another study showed, using a multiplex PCR-reverse line blot assay, that 48% of first voided urine specimens from women attending sexual health clinics in Australia were positive for U. parvumand 25% for U. urealyticum [17]. In the study by Tang et al., 36% of genitourinary swabs collected from hospitalized males and females in China were positive for U. parvumand 8% for U. urealyticum [10]. Finally, in study by Vancutsem et al., 44% of lower genital tract specimens obtained from healthy women at their first prenatal visit in Belgium were positive for U. parvumand 10% for U. urealyticum [11]. Despite different geographic locales and clinical status, these numbers are strikingly similar.In addition to the advantage of speed, the described assays overcome the challenges of detection of these organisms by culture. Although culture is considered a gold standard method (and was so considered in this study), colonial identification is challenging and subjective because it is done using the human eye and a dissecting microscope. Artifacts may be misidentified as colonies, yielding false-positive results, or colonies may be overlooked, yielding false-negative results. Although PCR may be considered more technically complex, in a laboratory where technologists are familiar with PCR, this approach is more user-friendly (and generalizable among assays for various microorganisms) than culture.The described assays may be useful for investigating epidemiology and pathogenesis of infections withU. parvumand urealyticum [2, 18]. Although extra-genital specimens were not tested, the described M. hominisassay may be useful to detect extra-genital M. hominisinfections [7].



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*Source: 168742-2013-03-11.xml*