Strand Displacement Amplification for the Diagnosis of Chlamydia by mikeholy

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									Presented at the 100th General Meeting of the American Society
for Microbiology, Los Angeles, CA 2000.




                      Strand Displacement Amplification for the Diagnosis of
                         Chlamydia and Gonorrhea in North Queensland
                                                                 M. AMADIO AND W.J.H. MCBRIDE
                                                       Cairns Base Hospital, Cairns, Queensland, Australia



      INTRODUCTION
s The diagnosis of Chlamydia trachomatis and
Neisseria gonorrhoeae infections using non-invasive
specimens by nucleic acid amplification has been a major
public health advance. The QHPS laboratory in Cairns has
been using the Roche Amplicor since the beginning of 1998.
About 800 tests per month are processed. The recognition
of false positive gonorrhoea results in specimens containing
N. subflava and N. cinerea prompted our use of the
16s rDNA confirmatory assay. We have subsequently
confirmed that false positive N. gonorrhoeae PCR results are a
                                                                                                                                                    METHODS
major problem in our population. Positive PCR results could
                                                                                   s The Roche Cobas Amplicor CT/NG assay, the 16s rDNA
not be confirmed in 32.4% of samples (unpublished data).                           N. gonorrhoeae confirmatory assay and the Becton Dickinson
The need for confirmatory testing has meant that positive                          ProbeTec ET assay were all performed according to
                                                                                   manufacturer protocols.
results are delayed. Other problems that have been observed                           Specimens were included in the evaluation if duplicate swabs
with the PCR method include the occurrence of samples that                         were received or urine volume was sufficient for both tests.

are inhibitory for PCR. An alternative nucleic acid amplification
method utilizing Strand Displacement Amplification (Becton
                                                                                                            Linear SDA                                                      Linear SDA
Dickinson ProbeTec) was evaluated in our laboratory                                         5'
                                                                                                                                           5'
                                                                                                                                                                5'
                                                                                      SDA Primer                                                                                                          5'
                                                                                                                             Target DNA

in May/June 2000.
                                                                                                 Restriction Eneyme                                                     Elongation of DNA strands by polymerase
                                                                                                 Recognition Site
                                                                                                 (Bsob I)




                                                                                                            Linear SDA                                                      Linear SDA
                                                                                            5'                                                                  5'
                                                                                                                                          5'                                                               5'




                                                                                                       Restriction enzyme "nicks" one strand


                                                                                         Unable to cut through both strands due to modified dC TP

                                                                                                                                                              Polymerase begins          New strand is "displaced"
                                                                                                                                                              elongation again           as elongation proceeds




                                                                                                            Linear SDA
                                                                                           5'
                                                                                                                                         5'




                                                                                                                                     New copies
                                                                                                                                     can serve as
                                                                                                                                     another template

REFERENCE                                                                             Nick and elongation
                                                                                      process repeats
1   Farrell, D.J. 1999. Evaluation of AMPLICOR
    Neisseria gonorrhoeae PCR using cppB Nested
    PCR and 16sr RNA PCR. J. Clin. Micro 37 (2) 386-390
                    SAMPLES
s A total of 385 samples were tested for
N. gonorrhoeae by both methods, 380
of these were also tested for Chlamydia.
The numbers for various specimen types
were:
        Swabs – 83
        Routine specimens – 369
        Urine – 302
        Stored, frozen or referred – 16



                                                                     RESULTS


              Chlamydia Results                              N. gonorrhoeae Results                   Repeat testing requirements for
                          PCR                                          PCR                                 PCR and SDA assays –
               +           –           NR                       +       –      NR
                                                                                                        Total No. of tests performed
        +      68          1           3    72          +       53     0        1      54
                                                                                                        CT/NG Multiplex PCR        493
  SDA




                                                  SDA




        –      2          290          12   304         –       1      313     13      327                                               618
                                                                                                    16s rDNA Confirmatory assays   125
        NR     1           2           1    4           NR      0      2        1      3

               71         293          16   380                 54     315     15      384             CT/NG Combined SDA          420   420

(NR = No result due to inhibition)



   False positive N. gonorrhoeae
              PCR tests
                                                                                             CONCLUSIONS
• 74 samples were positive in
  multiplex PCR
                                                                        The BD ProbeTec ET had several advantages over the Roche
• 54 of these samples were confirmed
                                                                      Cobas Amplicor for our patient population.
  positive in 16s rDNA assay                                          s Specimens containing N. gonorrhoeae are detected accurately
• 20 samples were negative                                              without the need for confirmatory testing, thus
• 27% FALSE POSITIVE RATE
                                                                      s There is a marked improvement in the turnaround time
                                                                        of positive results for N. gonorrhoeae
                                                                      s The total number of tests performed to achieve final results
   Prevalence of STI’s in the                                           is significantly fewer and should result in
random subpopulation (N=369)
     assessed by ProbeTec                                             s Cost savings
 • C. trachomatis        19%                                          s There were fewer inhibited samples (7 vs 31)
 • N. gonorrhoeae        12.7%                                        s There were fewer pipetting steps and the test was
 • Both                  5.1%                                           considered easier to learn and perform.



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