|Year : 2016 | Volume
| Issue : 4 | Page : 560-561
Gonococcal opa gene as a diagnostic target for nucleic acid amplification tests in Indian Population
R Verma, N Mahajan, S Sood
Department of Microbiology, All Institute of Medical Sciences, New Delhi, India
|Date of Submission||29-Sep-2015|
|Date of Acceptance||12-Sep-2016|
|Date of Web Publication||8-Dec-2016|
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Verma R, Mahajan N, Sood S. Gonococcal opa gene as a diagnostic target for nucleic acid amplification tests in Indian Population. Indian J Med Microbiol 2016;34:560-1
|How to cite this URL:|
Verma R, Mahajan N, Sood S. Gonococcal opa gene as a diagnostic target for nucleic acid amplification tests in Indian Population. Indian J Med Microbiol [serial online] 2016 [cited 2017 Feb 24];34:560-1. Available from: http://www.ijmm.org/text.asp?2016/34/4/560/195361
The switch from conventional to molecular methods for the laboratory diagnosis of gonorrhoea is inspired by difficulties associated with cultivation coupled with constraints with regard to its sensitivity. In an endeavour to keep pace, we have developed an in-house polymerase chain reaction (PCR) targeting the opa gene of Neisseria gonorrhoeae using self-designed primers (GenBank accession no. for primers: PUID 9716120 SNUM 2706 Ng_opa). The sensitivity, specificity, positive predictive value and negative predictive value for this assay were found to be 100% (95% confidence interval [CI] 92.5–100), 97.9% (95% CI 95.6–99.1), 89.5% (95% CI 79.1–95.3) and 100% (95% CI 98.6–100), respectively. We have been using this as the supplemental assay in conjunction with 16S ribosomal PCR for the detection of N. gonorrhoeae in urogenital samples. This is in compliance with 2002 CDC guidelines that prescribe routine repeat testing of all positive specimens. Recent CDC guidelines (2014) recommend retesting a positive genital tract specimen with an alternate target assay, primarily when nucleic acid amplification test used detects non-gonococcal Neisseria species. In light of this advisory, we wanted to further assess the specificity of our assay. Although a few strains of non-Neisseria N. gonorrhoeae sp. were included during standardisation studies, we went a step further to check the specificity of our PCR against a large panel of well-characterised commensal Neisseria sp. These included Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria subflava, Neisseria polysaccharea and Neisseria sicca. In addition, a phenotypically similar organism, Moraxella catarrhalis, which inhabits the upper respiratory tract, was included in the present investigation. [Figure 1] shows that our primers detected only gonococcal DNA, thereby testifying that our assay is a suitable 'stand-alone' test that can be adopted by laboratories equipped with a conventional PCR machine. Further, it may be relevant for use in specimens from anatomic sites such as oropharynx.
|Figure 1: Polymerase chain reaction results of opa gene. Lane 1: Blank, Lane 2: Control WHO F, Lane 3: 100 bp ladder, Lane 4: Neisseria gonorrhoeae ATCC 49226, Lane 5: Neisseria cinerea THO 148D10, Lane 6: Neisseria sicca ATCC 29193, Lane 7: Neisseria flavescens WARB 148D7, Lane 8: Neisseria lactamica KIRK 148E4, Lane 9: Neisseria sicca 1316 148E5, Lane 10: Neisseria mucosa BLEA 148E6, Lane 11: Neisseria subflava ELFO 148E10, Lane 12: Neisseria polysaccharea 33125 100F10, Lane 13: Moraxella catarrhalis NCTC11020 200J3, Lane 14: Neisseria sicca (clinical isolate), Lane 15: Neisseria sicca (clinical isolate), Lane 16: Neisseria sicca (clinical isolate)|
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Gonococcus has a plastic genome that allows alteration in its genotypic characteristics leading to not only false-positive results but false-negative results too in some gonococcal populations. It was earlier believed that by targeting a multicopy gene like opa, the chances of false-negative results due to sequence variation diminish because even if one copy mutates, there will be others that can be detected. However, a report to the contrary  has put us on guard. To scrutinise the stability of the target, we extracted DNA from 39 clinical isolates of N. gonorrhoeae from New Delhi and 22 clinical isolates obtained from across the country in the last 2 years. The presence of 188 bp amplicon in all affirms the conservation of the same among our local isolates, thereby offering a real potential for diagnostic use for Indian setting.
This is an inexpensive test and can be used in partnership with currently recommended syndromic management algorithms to improve the sensitivity and specificity of the approach. Such a strategy will be particularly invaluable in women and contribute to disease control.
The authors gratefully acknowledge Professor Monica M Lahra, Director & Dr. Athena Limnios, Quality Officer, Neisseria Reference Laboratory and WHO Collaborating Centre for STD, SEALS Microbiology, The Prince of Wales Hospital, Australia for presenting commensal Neisseria sp. to our laboratory.
Financial support and sponsorship
The work was supported by grant no. BT/PR11930/MED/29/118/2009 from the DBT, Ministry of Science and Technology, Government of India.
Conflicts of interest
There are no conflicts of interest.
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