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COMPARISON OF APTIMA COMBO 2, AMPLICOR AND
PROBETEC FOR THE DIAGNOSIS OF C. TRACHOMATIS
(CT) AND N. GONORRHOEAE (GC) IN SUREPATH L-PAP
SAMPLES 1M. Chernesky, 1D. Jang, 1M. Smieja,
2J. Kapala, 2C. Doucette, 2J. Sumner, 3R. Ewert,
3D. MacEachern, 3C. Pritchard, 4C.
MacRitchie 1St Josephs Healthcare/McMaster
University, Hamilton, ON, 2Gamma Dynacare Medical
Laboratories, Brampton, ON, 3Evergreen Health
Centre, Toronto, ON, 4Hamilton Community Health
Centre, Hamilton, ON, CANADA
Figure 2. L-Pap Sample Preparation
Objectives SurePath L-Pap samples have not
received FDA clearance for CT or GC testing in
approved NAATs. Laboratories are testing
SurePath samples using validation protocols. We
compared APTIMA Combo 2 AC2, ProbeTec PT
and AMPLICOR AMP assays to detect CT and GC
from SurePath L-Pap samples. Methods A total of
296 women consented to collection of a cervical
swab CS and 2 L-Pap collection vials. In the
laboratory the 2 L-Pap samples were pooled to
ensure homogeneity before processing for Pap
cytology. The remaining L-Pap fluid was tested
for CT and GC using published protocols for the 3
assays a transferring l ml into specimen
transport media before testing 400 µl in AC2
Gen-Probe Inc. b placing 500 µl into 2 ml of
PT diluent then testing 150 µl in PT Becton
Dickinson c pipetting 500 µl into a
centrifugation procedure leading to testing of 50
µl in AMP Roche. The CS was tested by AC2. To
compare the presence of analytes in L-Pap remnant
versus the gradient sample left after cytological
processing, both samples were tested by
AC2. Results The prevalence of CT was 11.2
33/296 and GC was 2.0 6/296. Two patients
were infected with both organisms. Twenty-five
75.7 of the 33 CS-positive women
CT-positives were positive in the L-Pap sample
in all 3 of the CT assays. The sensitivity of
AC2 was 97.0 32/33, 93.9 31/33 for AMP and
75.8 25/33 for PT. One CT-positive patient
was negative by all assays on the L-Pap remnant
but positive by AC2 on the gradient after
cytology processing. For GC, AC2 identified all
6 of the infections 100 while AMP and PT
missed one sensitivity 83.3. Conclusions
Using published protocols for CT and GC testing
of SurePath L-Pap samples demonstrated very high
sensitivity and specificity for the AC2 and AMP
assays. The protocol for PT testing of L-Pap
samples used in this study can not be
recommended. The remnant sample was slightly
better than the gradient for CT and GC testing by
AC2.
REVISED ABSTRACT
METHODS
Patient recruitment 296 women (ages 15-29)
from April 2008-January 2009 attending health
centre/ OB/GYN clinic for routine care signed an
IRB approved informed consent to participate in
the LPTSP (Liquid Pap Trial-SurePath) study.
Study participants with previous antibiotics use
in the past 3 weeks and women pregnant past the
first trimester were excluded. Physician
collection Each collection package included
information/informed consent forms and collection
kits, labeled with an unique study identifier.
The physician collected three samples (Fig. 1)
SurePath L-Pap sample following an established
procedure using the Cervex-Brush and immersed
into the specimen vial, an APTIMA unisex swab
(Gen-Probe Inc.) followed by the second L-Pap
SurePath sample (see diagram). Samples were
shipped the same day to Gamma Dynacare Medical
Laboratories (GDC), Brampton site.
Laboratory specimen handling/testing The
cytology technologist removed only the two L-Pap
vials from the study package and carefully mixed
both vials to ensure homogeneity. Both samples
were divided evenly. One vial remained for Pap
cytology at GDC and the other placed into the
study package with the rest of the study samples
and shipped immediately to St. Josephs
Healthcare Infections Research Group (SJH), where
the L-Pap sample was prepared for AC2, PT and
AMP within 48 hours (Fig. 2). APTIMA Combo 2
(Gen-Probe Inc.) As described previously,
after vortexing the SurePath vial, 1mL of L-Pap
was transferred into the APTIMA STM tube. Both
the cervical swab STM and L-Pap processed STM
were tested in the DTS 400/ Leader HC system.
BD ProbeTec (Becton Dickinson) As described
previously, 0.5mL of L-Pap was transferred into
the BD specimen dilution tube, inverted 3-4x and
tested according to the manufacturers
instructions. AMPLICOR CT/NG (Roche
Diagnostics) The protocol was initially
published (Cytyc Corporation 2003 in conjunction
with Roche Molecular Diagnostic System) for the
PreserCyt ThinPrep medium. Briefly, 0.5mL of
L-Pap was centrifuged at 12,500 g for 10 min,
decanted and the pellet was treated with CT/NG
lysis buffer (Roche) and further resuspended into
specimen diluent. A 50µl volume was used in the
CT/NG mastermix. The amplification and detection
with micro-well plates (MWP) was performed
according manufacturers instructions.
Sensitivity and Specificity calculations
Calculations were made based on the CS being
positive or negative. Confidence intervals were
calculated using confidence intercal analysis
software (version 2.1.2, 2004 T. Bryant,
University of Southampton, UK).

AMP CT/NG
PT
AC2
Cervical swab into APTIMA STM
0.5 ml L-Pap into Eppendorf tube
0.5ml L-Pap into BD Specimen Dilution tube
1ml L-Pap into APTIMA STM
RESULTS
Table 1. Testing profiles for 33 infected
patients
Table 2. Sensitivity and Specificity of AC2, AMP
and PT on SurePath L-Pap samples
INTRODUCTION
Liquid based cytology for the detection of
cytolological abnormalities leading to cervical
cancer has demonstrated advantages over
conventional smear preparation and has been
adopted by many laboratories. Currently there are
three liquid based cytology mediums cleared by
the US Food and Drug Administration for
liquid-based Pap (L-Pap) cytology PreservCyt
ThinPrep (Hologic), SurePath (BD
Diagnostics-TriPath) and Cytotek MonoPrep
(Monogen). All use broom, brush or spatula
sampling of the cervix placed into their
proprietary transport media. AC2 and AMP have
FDA clearance for the use of ThinPrep L-Pap for
C. trachomatis (CT) and N. gonorrhoeae (GC)
testing. Our laboratory as well as others have
been interested in using the SurePath L-Pap
samples for testing CT/GC by APTIMA Combo 2
(Gen-Probe Inc.), BD ProbeTec (Becton Dickinson)
and AMPLICOR CT/NG (Roche Diagnostics) assay.

CT

GC Assay Sensitivity
Specificity Sensitivity
Specificity
(95 C.I) (95 C.I) (
95 C.I) (95 C.I)
AC2 97.0 (32/33) 100
(263/263) 100 (6/6) 100
(290/290)
(84.2-99.5) (98.6-100)
(61.0-100) (98.7-100)
AMP 93.9 (31/33) 99.6
(262/263) 83.3 (5/6) 98.9
(287/290)
(80.4-98.3) (97.9-99.9)
(43.6-97.0) (97.0-99.6)
PT 75.8 (25/33) 100
(263/263) 83.3 (5/6) 100
(290/290)
(59.0-87.2) (98.6-100)
(43.6-97.0) (98.7-100)

Figure 1. Physician instructions for specimen
collection
1. 2.
3.
CONCLUSIONS

• The prevalence of infection in our study
  population was 11.2 (33/296) for CT and 2
  (6/296) for GC based on CS.
• SurePath L-Pap testing demonstrated excellent
  sensitivity and specificity with the APTIMA Combo
  2 and AMPLICOR assays.
• 24.2 of the CT positives were missed using the
  ProbeTec protocol for SurePath samples.
• Presented at the 18th International Society for
  STD Research (ISSTDR/BASHH) June 28thJuly 1st,
  2009, London, UK

OBJECTIVE
Insert the central bristles into the
endocervical canal deep enough to allow the
shorter bristles to fully contact the ectocervix.
Push gently, and rotate the broom in a clockwise
direction five times.
To compare the performance of APTIMA Combo 2
(AC2), BD ProbeTec (PT) and AMPLICOR CT/NG (AMP)
on SurePath L-Pap samples for the diagnosis of CT
and GC.