376 J Clin Pathol 1991;44:376-379 Identification of 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 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 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 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 of a prospective study of identification of 400 isolates ofNgonorrhoeae using Neisstrip, CTA, and Phadebact monoclonal GC identification systems

Neisstrip CTA sugars

Identity by J Clin Pathol: first published as 10.1136/jcp.44.5.376 on 1 May 1991. Downloaded from Number of isolates fl-G PA GGA Glucose Maltose Sucrose Phadebact API 20NE Ngonorrhoeae 92 - + - + -(24 h) + 9 + - + -(48 h) + 2 - + - + 295 + - + Organisms misidentified as Ngonorrhoeae 2 - + - Moraxella spp ,B-G: ,B-galactosidase, PA: prolyl aminopeptidase, GGA: gammaglutamyl aminopeptidase, CTA: cystine trypticase agar.

CTA TEST Discussion Organisms were inoculated on to serum-free The pressure for accuracy and speed in the cystine trypticase agar (CTA) media contain- identification of N gonorrhoeae has led to the ing appropriate sugars, which when degraded development ofseveral commercial kits,2 which during incubation at 37C for 24-48 hours, perform well but add to cost and require produced acid, and thus a change in the colour refrigeration. In the past the identification of of the pH indicator present in the media.3 In the organism for legal purposes required the this study the media were incubated for 48 production of acid from glucose but not from hours and results compared with those noted other sugars. False results do occur and further after 24 hours. Flynn and Waitkins' test was tests may be required.6 similarly performed and interpreted. The change in the colour ofthe pH indicator in the medium is slow, difficult to see, and is SUPEROXOL TEST affected by dissolved carbon dioxide, so this Using the method of Young et al,5 a colony to inaccuracy is not surprising. be tested was rubbed on to a glass slide and a In the study of the culture collection strains, drop of 30% (w/v) hydrogen peroxide added. which included bacteria known to be difficult to A positive test was indicated by the produc- identify, none of the organisms was incorrectly tion of large numbers of bubbles within two to identified by both Phadebact and Neisstrip nor three seconds. by both Phadebact and Gonochek. The repor- ted false positive cross reaction of N cinerea, N Results lactamica, N meningitidis and Branhamella The four methods used to identify the culture catarrhalis using the pooled monoclonal collection isolates all performed well (table 2). antibody products in the identification of N http://jcp.bmj.com/ The organism that produced PA but not GGA, gonorrhoeae suggest that further tests may be utilised glucose and maltose, but was required to be sure of the identity of a strain. Phadebact negative, was identified as a non- This has not been shown at this time with the groupable N meningitidis type 14, subtype Phadebact monoclonal GC test which has been p 1. 15 by the Meningococcal Reference reported as 100% sensitive and specific.7 N Laboratory at Manchester Public Health cinerea is exceedingly uncommon in genital Laboratory, which also confirmed our results. specimens but may give a positive sugar utilisa- on September 29, 2021 by guest. Protected copyright. Of the 400 isolates identified in the prospec- tion reaction with glucose (and not other tive trial (table 3), all gave consistent results sugars). Its identification is therefore difficult. with both the Neisstrip and the Phadebact Neisseria lactamica, however, is clearly differen- tests, except for two species ofMoraxella which tiated from N gonorrhoeae by both rapid had been isolated on gonococcal selective enzyme tests and the CTA test. The same time media, and two strains ofNgonorrhoeae, which (15 minutes) is required to use the Neisstrip as failed to give positive PA tests. Of 10 organisms the Phadebact test, which may allow them to be identified using sugar degradation methods carried out concurrently. during the period of the study, two N menin- The lack of false negative or false positive gitidis strains were misidentified but gave results when using both Neisstrip and correct results when using both Phadebact and Phadebact monoclonal GC in the culture Neisstrip. The superoxol test gave results con- collection study allowed us to validate the use sistent with Neisstrip and Phadebact, except of further identification methods in the pros- that of 29 isolates from the selective media that pective study only if the results of these tests were not N gonorrhoeae, four gave a positive disagreed. The finding that the only two organ- superoxol result (table 4). isms in which this occurred were Moraxella spp is not surprising in that these may produce PA and may be found in the upper respiratory Table 4 Superoxol test results on 233 oxidase positive coloniesfrom separate genital swabs cultured on modified tract. To qualify further the occurrence of PA Thayer-Martin gonococcal selective agar in isolates of the genus Moraxella a further three strains were tested and found to be Organism identity by Superoxol Superoxol negative. Phadebact GC and Neisstrip tests positive negative The advantages of chromogenic substrate Neisseria gonorrhoeae 204 0 tests have been reported.8 The ability to give an Non-N gonorrhoeae 4 25 indication of the identity of an organism that is Rapid identification ofNgonorrhoeae 379

not N gonorrhoeae is available with Gonochek medium, when tested by the superoxol test and II (although the slow production of colour by Neisstrip, gave a false positive rate which we some strains of N meningitidis is a problem4), estimate to be less than 0 1%. Use of these two and with a system similar to Neisstrip.8 Organ- tests would require that ifonly one was positive isms that did not produce any colour change then the result should be checked by other J Clin Pathol: first published as 10.1136/jcp.44.5.376 on 1 May 1991. Downloaded from were considered to be Branhamella catarrhalis methods. in these studies, but it is clear that this pattern We conclude that Neisstrip is a convenient, can be produced by other organisms,8 includ- rapid system for the identification of N gonor- ing the two Moraxella spp in this study. We feel rhoeae from selective media for the isolation of it would be unwise to identify B catarrhalis gonococci, and that it may be of use in the using Neisstrip or Gonochek II, and their use identification of other pathogenic Neis- to identify N meningitidis should only be in seriaceae. When used alone in the circum- conjunction with further tests. They would be stances that we describe it has a sensitivity of useful, however, to help identify aberrant 99%, a specificity of 95% giving a positive strains of meningococci that are maltose predictive value of 99%. In combination with negative.5 Nsubflava biovarperflava or Kingella the superoxol test it is exceedingly accurate. denitrificans do not occur commonly in pharyn- geal samples, but their ability to produce PA 1 Knapp JS. Historical perspectives and identification of may cause them to be misidentified by both Neisseria and related species. Clin Microbiol Rev Neisstrip or Gonochek II as N gonorrhoeae 1988;1:415-31. 2 Dillon JR, Carballo M, Pauze M. Evaluation of eight from gonococcal selective media. Hence, the methods for identification ofpathogenic Neisseria species: use of either alone to test colonies from the Neisseria-Kwik, RIM-N, Gonobio-Test, Minitek, Gono- chek II, GonoGen, Phadebact Monoclonal GC OMNI pharynx may prove inadequate. Similarly, the Test and Syva Micro Trak Test. J Clin Microbiol cross reactions of other monoclonal antibody 1988;26:493-7. 3 Morello JA, Janda WM, Bohnhoff M. Neisseria and Bran- based tests with pharyngeal Neisseriaceae2 may hamella. In: Lennette EH, et al, eds. Manual of clinical suggest that these too should not be used alone microbiology, 4th edn. Washington: American Society for Microbiology, 1985:176-92. in identification ofgonococci from the pharynx. 4 Wood IA, Young H. Identification of pathogenic Neisseria Pharmacia's Monoclonal GC Test, however, is by enzyme profiles determined with chromogenic sub- strates. Med Lab Sci 1986;43:24-7. not directly comparable with these other tests.2 5 Young H, Harris AB, Tapsall JW. Differentiation of gono- The superoxol test requires experience to coccal and non-gonococal neisseriae by the superoxol test. Br J Vener Dis 1984;60:87-9. differentiate positive and negative results but 6 Davies AJ, Jephcott AE. Bacteriology ofthe genital tract. In: gave results consistent with Neisstrip and with Hawkey PM, Lewis DA, eds. Medical bacteriology. Edin- burgh: Churchill Livingston, 1989:71-89. Phadebact Monoclonal GC in 98% of organ- 7 Young H, Reid KG. Immunological diagnosis ofgonococcal isms tested in the prospective study. It is rapid, infection. In: Young H, McMillan A, eds. Immunological diagnosis of sexually transmitted diseases. New York: and cheap, but shows some positive results Marcel Dekker Inc, 1988:98-9. with other Neisseriaceae, particularly N lac- 8 Janda WM, Sobieski V. Evaluation of a ten-minute tamica, Nmeningitidis, and B catarrhalis.5 Non- chromogenic substrate test for identification ofpathogenic

Neisseria species and Branhamella catarrhalis. Eur J Clin http://jcp.bmj.com/ gonococcal isolates from gonococcal selective Microbiol Infect Dis 1988;7:25-9. on September 29, 2021 by guest. Protected copyright.