Center for Emerging Infectious Diseases

1
BD ProbeTec ET System for the Detection of
Mycoplasma pneumoniae from Clinical Respiratory
Specimens
Center for Emerging Infectious Diseases Univ. of
Iowa College of Public Health 200 Hawkins Dr.,
C21K GH Iowa City, IA 52245 Tel (319)
384-5008 Fax [phone] www.public-health.uiow
a.edu/CEID
Gregory C. Gray1, Lynn B. Duffy2, Charlotte A.
Gaydos3, Christine C. Ginocchio4, William LeBar5,
Judy Lovchik6, Margaret R . Hammerschlag7, Tony
Mazzulli8, Richard Rothman3 1Center for Emerging
Infectious Diseases, University of Iowa College
of Public Health, Iowa City, IA 2University of
Alabama at Birmingham, Birmingham, AL 3Johns
Hopkins University, Baltimore, MD 4North Shore
Long Island Jewish Health System Laboratories,
Lake Success, NY 5Hospital Consolidated
Laboratories Providence Hospital, Southfield,
MI 6University of Maryland Medical Center,
Baltimore, MD 7SUNY Downstate Medical Center,
Brooklyn, NY 8Mount Sinai Hospital Department of
Microbiology, Toronto, Ontario
Revised Abstract
Table 2. BD ProbeTec ET lower respiratory MP
assay vs. culture
Study purpose To evaluate the BD ProbeTec ET
Mycoplasma pneumoniae Amplified DNA Assay, which
is part of a group of assays for atypical
pneumonia including Legionella pneumophila, and
Chlamydiaceae family (Not for sale in the U.S.,
these assays are under FDA review).
Background Mycoplasma pneumoniae (MP) is a
frequent cause of acute respiratory infection,
especially pneumonia, and may lead to severe
morbidity and death. In some populations, MP may
cause as much as 20 of community acquired
pneumonia (CAP). MP infections are difficult to
detect culture and serology lack sensitivity,
while molecular techniques are often complex. We
evaluated a new Strand Displacement Amplification
(SDA) assay on the BD ProbeTec ET System (BD
Diagnostics) for the detection of MP among
patients with CAP. Methods Patients one year
old and greater with radiographic evidence of
pneumonia were enrolled at 7 medical sites
between October 2002 and July 2003. Lower
respiratory specimens (LRS) and throat swabs (TS)
were collected from patients who granted informed
consent. Specimens were tested for evidence of MP
by culture, PCR, and the BD ProbeTec ET MP Assay.
The University of Alabama at Birminghams
mycoplasma laboratory performed MP culture of
throat swabs and PCR procedures using previously
published techniques. The MP Assay employs
simultaneous amplification and real-time
detection of target DNA and an internal control
using amplification primers and fluorescently
labeled detector probes. LRS and TS panels spiked
with high and low levels of MP were tested in the
MP Assay for reproducibility at 3 sites.
Results During the nine-month study, 5 (1.6)
of 316 enrolled patients had some evidence of MP
infection. The specificity of the MP Assay
compared to culture was 99 for both LRS and TS.
Specimens that were discrepant (MP Assay vs.
culture) were further studied with PCR. All 3 MP
Assay positive but culture negative specimens
(one LRS and 2 TS) were positive for MP by PCR.
Two specimens (LRS and TS from the same patient)
that were MP Assay negative but culture positive
were both found to be PCR negative. The LRS
reproducibility with spiked samples was 92 for
the low level and 100 for the high level and
negatives. The TS reproducibility with spiked
samples was 100 for all three levels.
Conclusions These data suggest that the MP
Assay has excellent specificity, reproducibility,
and good correlation with PCR for detection of
MP.
Materials Methods
PCR negative (patient H1013) PCR positive
(patient T021)

• Subjects Patients 1 year of age with signs and
  radiographic evidence of pneumonia were enrolled
  from 7 medical sites patients with antibiotic
  therapy lt14 days
• Specimens After informed consent, patients
  permitted the collection of
• Lower respiratory specimen (LRS) - sputum
  (expectorated, induced, endotracheal suction or
  collected during bronchoscopy)
• Throat swabs (TS)
• Sera (patients 5-13 years only) acute sera only
• Laboratory methods
• All patients LRS and TS evaluated with BD
  ProbeTec ET MP assay. TS collected and
  expressed in SP4 medium for MP culture and MP PCR
  (University of Alabama)
• Children 5-13 years Serological IgM test for MP

Table 3. BD ProbeTec ET throat swab MP assay vs.
culture
PCR negative (H1013) PCR positive
(patients I022 and N106)
Table 4. BD ProbeTec ET MP lower respiratory
assay - reproducibility
Results

• A total of 5 (1.6) of the 316 enrolled pneumonia
  patients had evidence of MP infection by BD
  ProbeTec ET MP assay
• The specificity of BD ProbeTecET MP assay was
  99 for LRS and TS
• Discrepant (BD ProbeTecET vs. culture) specimens
  were MP PCR positive.
• Two specimens (LRS and TS from the same patient)
  that were BD ProbeTecET negative but culture
  positive were all found to be PCR negative.
• The BD ProbeTecET LRS overall reproducibility
  was 92 for the low level, 100 for the high
  level and negatives.
• The BD ProbeTecET TS overall reproducibility was
  100 for all levels.

Introduction
One sample excluded from analysis due to an
operator pipetting error.

• The BD ProbeTec ET System uses a proprietary
  real time molecular amplification technology,
  Strand Displacement Amplification (SDA), in a
  closed reagent system that does not require
  unidirectional workflow or separate work areas.
• Using simple, dried down, ready-to-use reagents
  and smooth workflow, the system assay time is 1
  hour for up to 94 specimens.
• In the United States, this system has three FDA
  cleared assays
• Chlamydia trachomatis (CT) assay
• Chlamydia trachomatis/ Neisseria gonorrhoeae
  (CT/GC) assay
• Legionella pneumophila (LP) assay

Table 5. BD ProbeTec ET MP throat swab -
reproducibility
Table 1. Data from specimens that had at least
some laboratory evidence of MP
One sample excluded from analysis due to an
operator pipetting error.

• Mycoplasma pneumonia (MP)
• Frequent cause of community acquired pneumonia
• Treatable but difficult to detect through
  currently available rapid tests, culture, and
  serology

Conclusions
These data suggest that the BD ProbeTec ET MP
Assay has excellent specificity, excellent
reproducibility, and may be as sensitive as PCR
in detecting MP.
MP Mycoplasma pneumoniae TS Throat swab LRS
Lower respiratory specimen