376 J Clin Pathol 1991;44:376-379 Identification of Neisseria gonorrhoeae using the Neisstrip rapid enzyme detection test J Clin Pathol: first published as 10.1136/jcp.44.5.376 on 1 May 1991. Downloaded from S F Dealler, K R Gough, L Campbell, A Turner, P M Hawkey Abstract glutamyl aminopeptidases. This test was com- A rapid enzyme activity strip test (Neis- pared with sugar utilisation using cystine strip, Lab M Ltd, Bury) was compared trypticase agar (CTA),' Phadebact Mono- retrospectively with Phadebact Mono- clonal GC coagglutination (Pharmacia Diag- clonal GC coagglutination (Pharmacia nostics AB, 75182, Uppsala, Sweden), and the Diagnostics, Uppsala, Sweden), cystine Gonochek II rapid enzyme detection system trypticase agar sugar utilisation (CTA), (J W Turner Ltd, Liverpool). These kits were and Gonochek II ( W Turner, Liverpool) evaluated using a culture collection from the enzyme methods for identification of 95 Gonococcus Reference Unit which included Neisseria spp and related species. These strains of N gonorrhoeae known to give had been previously identified using stan- aberrant results in conventional identification dard methods and included 29 that had tests. We also prospectively evaluated the given aberrant results. Neisstrip identi- Neisstrip, Phadebact Mohoclonal GC test, fied correctly all but two, including nine CTA test and the superoxol test against incorrectly identified by Phadebact and unselected isolates of presumptive gonococci 18 erroneously identified using CTA in clinical samples from four genitourinary sugars. Results were similar to those medicine departments, and also strains obtained with Gonochek II. After this a referred to the Gonococcus Reference Unit prospective study was performed testing from around the United Kingdom. 400 oxidase positive isolates derived from clinical samples cultured on gono- coccal selective medium. Two organ- isms, both Moraxella spp, were in- Methods correctly identified as N gonorrhoeae by Ninety three strains of Neisseria spp and the Neisstrip. The superoxol test, when related species that had been isolated from used with either the Phadebact or Neis- clinical samples taken in genitourinary clinics http://jcp.bmj.com/ strip tests, maintained 100% sensitivity or referred to the Gonococcus Reference Unit and specificity. were fully identified using standard tech- The Neisstrip is a rapid, economic test niques.3 These isolates would grow on that is accurate and easy to interpret. It modified New York City medium' and were may be used alone or in conjunction with considered to be representative of organisms a superoxol test or a coagglutination test, that could be isolated on gonococcal selective which is relatively accurate but more media in clinical laboratories; some, however, on September 29, 2021 by guest. Protected copyright. expensive, and found by some technical had given aberrant results in identification staff to be more difficult to interpret. tests. Each of the 93 organisms was tested using the Neisstrip, Gonochek II, Phadebact Monoclonal GC Test, and sugar utilisation The identification of Neisseria gonorrhoeae by method (CTA sugars).3 The identity of one sensitive, precise, and rapid techniques is meningococcus had to be confirmed by the necessary for the correct diagnosis and Meningococcal Reference Laboratory, Man- appropriate treatment of patients with gonor- chester, England. rhoea. For many years the sugar utilisation For the prospective study clinical samples Department of tests have been the preferred method, but the from the Department of Genitourinary Microbiology, advent of rapid methods for detecting Medicine at Leeds General Infirmary, Leeds, University of Leeds, antigens or enzyme activity of the bacterium and St Luke's Hospital, Bradford, were cul- Leeds LS2 9JT S F Dealler have permitted faster results to be obtained.' tured on modified Thayer Martin agar (Oxoid P M Hawkey The falling number of positive cultures for N Ltd, Basingstoke). Isolates, if found to be Gonococcus Reference gonorrhoeae in recent years has increased the oxidase positive Gram negative cocci, were Unit, PHLS, Bristol requirement for tests which are highly tested using the Phadebact Monoclonal GC K R Gough as individual workers' Test, the Neisstrip, and, at Leeds, the A Turner reproducible laboratory experience of isolating and identifying N gon- superoxol test. Any samples for which the Department of Microbiology, orrhoeae declines. An increasing number of results of the first two tests disagreed were Bradford Royal tests and kits have consequently been appear- further identified using Flynn and Waitkins' Infirmary ing on the market, particularly in the United serum-free sugar media (Difco, East Molesey) L Campbell States of America.2 using standard methods.3 Isolates of N gonor- Correspondence to: the evaluation of a rhoeae that were referred to the Gonococcus Dr S F Dealler We describe Neisstrip, 15 minute test which detects the bacterial Reference or isolated in their two Accepted for publication Unit, 6 December 1990 enzyme f,-galactosidase, and prolyl and y- genitourinary clinics during the same period Rapid identification ofNgonorrhoeae 377 Table 1 Biochemical characteristics ofNeisseria capable ofgrowth on gonococcal former and a purple colour in the latter were selective media after Knapp' taken as positive results. Most strains of N gonorrhoeae produce PA but not GGA. Other Acidfrom members of the genus Neisseria give the com- J Clin Pathol: first published as 10.1136/jcp.44.5.376 on 1 May 1991. Downloaded from Species Glucose Maltose Lactose P-G PA GGA Superoxo1l bination of results shown in table 1. Ngonorrhoeae + - - + - + N meningitidis + + - + - PHADEBACT MONOCLONAL GC TEST N cinerea + - - + B catarrhalis - This test relies on the presence of the major K denitrificans + - - + outer membrane protein (MOMP) protein I on the surface of the bacterium. This protein fl-G: ,B-galactosidase, PA: Prolyl or hydroxy prolyl aminopeptidase, GGA: Gamma glut: -myl aminopeptidase. *This result may be weak and is not positive in all test systems. amyl is only found in strains of Ngonorrhoeae. It is detected by the coagglutination of a suspen- sion of non-viable Staphylococcus aureus cells were tested similarly but with Flynn and to which are attached a monoclonal antibody Waitkins' and CTA tests. which is specific for the MOMP. A light Bacteria that were identified as gonoc()cci suspension of the isolate in 0 5 ml of 0-9% using the Neisstrip but not by the ot:her (w/v) saline was placed in a test tube in a methods were further identified using the i\PI boiling water bath for five minutes. One drop 20NE kit (API Systems, Montalieu-Verviieu, of each Phadebact reagent was placed on a France). white card. Each was then mixed with a drop of the boiled extract. The suspensions were NEISSTRIP COLONY IDENTIFICATION gently rocked, and agglutination in one well A single colony was taken from the selec:tive but not the other within one minute was medium and inoculated on to each of the tlhree considered a positive reaction. chemically impregnated filter paper strips that are attached to a plastic strip. Each of the GONOCHEK filter papers was moistened with 10 4ul of dis- This system relies, as does Neisstrip, on the -tilled water, and the strip placed in a P'etri differential ability ofthose Neisseriaceae which dish and incubated at 37°C for 15 minuites. will grow on gonococcal selective medium to The dish also contained a 2 cm square piec:e of produce fl-G and two aminopeptidases.1 A damp blotting paper to prevent the strip ciry- heavy suspension of the organism was ing out. The development of a blue colouxron inoculated from the selective medium into a the fl-galactosidase (fl-G) strip was takeri to test tube containing chromogenic substrates indicate the presence of this enzyme, whicJh in for the enzymes. The tube was incubated for turn indicated that this organism could noit be 30 minutes at 37°C and the presence of N gonorrhoeae. If no blue colour appeared a colours in the test tube after this time or chemical developer was added to the priolyl indicated the presence absence of enzyme http://jcp.bmj.com/ aminopeptidase (PA) and the y-glutaimyl activity.4 Yellow was taken to indicate the aminopeptidase (GGA) strips. The formatiion, presence of GGA activity, blue fl-G, and red within 30 seconds, of a pink colour in the (after the addition of EY-20) PA. Table 2 Results of comparative identification of 93 bacterial isolates the culture collection of Gonococcal Reference Laboratory on September 29, 2021 by guest. Protected copyright. Neisstrip CTA sugars Phadebact Number of isolates fi-G PA GGA Glucose Maltose Sucrose monoclonal Gonochek Neisseria gonorrhoeae 9 - + - + - Red 4 + + Red Neisseria meningitidis 42 - - + + + - Yellow 6 - - + + - - Yellow 4 -- + - + - - Yellow 3 - - + - - Yellow 1 - - + + - - Yellow 1 - + + - - Red Neisseria lactamica 9 + - - + + - - Blue + - - + - - - Blue 1 - ±+ + + - - Blue Neisseria cinerea 2 - + - - - - - Red Branhamella catarrhalis 1 - + - - - - - Yellow 2 - - + - - - - Colourless 4 - - - - - - - Yellow Moraxella spp. 2 - - + - - - - Yellow 1 - - - -- - - Colourless ,B-G: (l-galactosidase, PA: prolyl aminopeptidase, GGA: gamma-glutamyl aminopeptidase, CTA: cystine trypticase agar. Gonochek colours: yellow and blue without addition of EY-20, red and colourless after the addition of EY-20. All isolates were capable of growth on gonococcal selective media with exception ofN cinerea. 378 Dealler, Gough, Campbell, Turner, Hawkey Table 3 Results