Download MAMEF POC Test for Detection of CT

Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) Point-ofCare Test for the Detection of Chlamydia trachomatis
Melendez JH1, Huppert J2, Hesse E2, Jett-Goheen M3, Quinn, N3, Gaydos CA3, Geddes CD1*
1Institute
of Fluorescence and Department of Chemistry and Biochemistry, UMBC, Baltimore, MD, USA
2Cincinnati children’s hospital Medical Center, Cincinnati, OH, USA
3Johns Hopkins Medical Institutions, Division of Infectious Diseases, Baltimore, MD, USA
*[email protected]
Background
Results
Chlamydia trachomatis (CT) is the most commonly reported sexually-transmitted
infections (STI). In 2011, there were over 1.2 million cases of chlamydia reported to the
CDC. This infection has serious sequelae among women including PID, tubal factor
infertility, chronic pelvic pain, and ectopic pregnancy. Chlamydia infections can increase
HIV transmission 3-5 fold.
Often persons who present to a clinic for STI testing never return to the clinic to receive
their STI test results. Thus the availability of a point-of-care test which can be resulted to
them immediately is highly desirable. In the present study, we report the results of a
blinded study evaluating the performance of two microwave-accelerated metal-enhanced
fluorescence (MAMEF) assays for detection of CT DNA. MAMEF combines the benefits
of low power microwave acceleration (MA) to rapidly accelerate biological reactions to
completion within seconds with those of metal-enhanced fluorescence (MEF) for
amplification of the fluorescence signal.
 The overall prevalence of CT in this sample set was 17.7%
 257 vaginal swabs from 245 subjects ranging in age from 14 to 22 years of age
 88.6% of subjects were African American and the majority of subjects with a
Chlamydia infection were African American (95.6%)
Methods
In the present study, detection of CT DNA is mediated by a two-step process. First, CT
cells from vaginal samples are rapidly lysed and the DNA fragmented using lysing
chambers composed of gold triangles deposited on glass slides and heated using
conventional microwave irradiation (Figure 1). After a centrifugation step, detection of
CT genomic DNA is carried in silver-covered wells known as fire-in-the-hole (Figure 1),
which have been shown to enhance the fluorescence signal. Detection of genomic DNA is
mediated by the complementary binding of two probes to the target sequence as shown in
Figure 2. For increased sensitivity and specificity, two sets of probes targeting the 16S
rRNA gene and the cryptic plasmid of CT were developed and tested. The flow chart
showing the sequence of events and timeline for a MAMEF-based CT test are shown in
Figure 1.
Table 1. MAMEF results vs. NAATs using vaginal swabs
MAMEF Assay
Elution of
vaginal swab
in distilled
water
Microwave
lysing
(35 seconds)
Centrifugation
(3 minutes)
Detection by
MAMEF
(< 5 minutes)
Data analysis
Sensitivity
(%)
Specificity
(%)
16S rRNA and cryptic
plasmid combined
73.3
92.9
16S rRNA
75.5
92.9
Cryptic plasmid
82.2
92.9
Table 2. Sensitivity and specificity of the MAMEF assays
Lysing chamber
Conclusions
Silver-covered disposable wells for
MAMEF-based CT detection
 Our lysing approach can effectively lyse cells and fragment DNA
in 35 seconds
Figure 1. Sequence of events and timeline for MAMEF-based CT detection
 Dry vaginal swabs are an effective collection method for MAMEF testing
 The cryptic plasmid MAMEF-based assay is more sensitive than
the 16S rRNA assay
 Specificity of both MAMEF assays is very high
 Our CT assay can specifically detect over 80% of cases in less than 10 minutes
 MAMEF is an ultra-fast and sensitive approach for detection of CT
Reference and acknowledgements
Zang Y, Agreda P, Kelly S, Gaydos C, Geddes CD. Development of a microwave-accelerated
metal –enhanced fluorescence 40 second, 100 cfu/mL point of care assay for the detection of
Chlamydia trachomatis. IEEE Trans BioMed Eng 58:781-784, 2011.
Figure 2. Probe-based CT detection using MAMEF
The authors would like to thank The Meyerhoff Graduate Fellowship Program, The Institute of
Fluorescence and the Department of Chemistry and Biochemistry, University of Maryland Baltimore
County for support. Financial support from the NIH / NIAID MARCE - Midatlantic Regional Center of
Excellence for Biodefense and Emerging Infectious Diseases (NIAID/NIH) 2 U54 AI057168-06 and
NIH/NIBIB 1U54 EB007958-4 are also gratefully acknowledged.
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