Real-time PCR detection of Neisseria gonorrhoeae susceptibility to penicillin

<span class="paragraphSection"><div class="boxTitle">Objectives</div>The objective of this study was to develop a real-time PCR assay targeting the gonococcal <span style="font-style:italic;">porB</span> gene (PorB-PCR) for predicting susceptibility of <span style="font-style:italic;">Neisseria gonorrhoeae</span> to penicillin. This complements a previously described PCR assay for detecting penicillinase-producing <span style="font-style:italic;">N. gonorrhoeae</span> (PPNG) developed by our laboratory (PPNG-PCR).<div class="boxTitle">Methods</div>The PorB-PCR assay was designed using six probes to characterize various combinations of amino acids at positions 101 and 102 of the PorB1b class protein, including the WT G101/A102 and mutant G101K/A102D, G101K/A102N and G101K/A102G sequences, as well as the PorB1a sequence. The ability of these sequences to predict penicillin susceptibility was initially assessed using 2307 <span style="font-style:italic;">N. gonorrhoeae</span> isolates from throughout Australia for which phenotypic susceptibility data were available. The assay was then applied to <span style="font-style:italic;">N. gonorrhoeae</span>-positive clinical specimens (<span style="font-style:italic;">n</span> = 70). Specificity was assessed by testing commensal <span style="font-style:italic;">Neisseria</span> strains (<span style="font-style:italic;">n</span> = 75) and <span style="font-style:italic;">N. gonorrhoeae-</span>negative clinical specimens (<span style="font-style:italic;">n</span> = 171).<div class="boxTitle">Results</div>Testing of the 2307 <span style="font-style:italic;">N. gonorrhoeae</span> isolates using PorB-PCR to detect G101/A102 and PorB1a sequences identified a total of 78.4% (61.2% and 17.2%, respectively) of penicillin-susceptible isolates with specificities of 97.4% and 99.3% and positive predictive values of 98.8% and 98.9%, where PPNG strains were simultaneously identified and excluded. Similar performance data were obtained when the PorB-PCR assay was applied to the <span style="font-style:italic;">N. gonorrhoeae-</span>positive clinical specimens. No false-positive results were observed for the <span style="font-style:italic;">N. gonorrhoeae</span>-negative samples and no cross-reactions were observed with the non-gonococcal species.<div class="boxTitle">Conclusions</div>When used in parallel with the previously described PPNG-PCR, the PorB-PCR approach has the potential to facilitate individualized treatment of gonorrhoea using penicillin.</span>

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