0095-1137/81/070089-05$02.00/0
Comparison of the Phadebact Gonococcus Test with the
Rapid Fermentation
Method
SUSAN L. FUTROVSKY,I* CHARLOTTE A. GAYDOS,2ANDJOHN . KEISER2
Departmentof Pathology, Walter ReedArny Medical Center,2 and Department of Bacterial Diseases, Walter Reed Army Institute of
Research,1
Washington, DC 20012Received7November 1980/Accepted17March 1981
The PhadebactGonococcus Test(PharmaciaDiagnostics, Piscataway, N.J.),a
coagglutination technique,wascomparedwiththerapidfermentation method of
Kelloggand Turner (D. S. Kellogg, Jr.,and E. M. Turner,Appl. Microbiol. 25:
550-552, 1973). A total of 93 organisms isolated on Martin-Lewis media were
determined tobe Neisseriagonorrhoeae basedon thefollowingcriteria: presence
ofgram-negative diplococci, oxidase positivity, and appropriate reaction in the
rapidfermentationmethod. These 93 isolates were then serologically tested with
the Phadebact test. The direct method was attempted on the first 46 N.
gonor-rhoeaeisolates.Difficultyininterpretingresults wasencounteredin39%.
There-after, the alternate method of boiling was instituted on an additional 47 N.
gonorrhoeae isolates, with 2 isolates producing noninterpretable results. All93
isolates were frozen for amaximumof 2 months in skim milk at-25°C. These
isolateswerethawed andretyped with the alternate boiling procedure, with 97%
being confirmed as N. gonorrhoeae. In addition, 33 Neisseria meningitidis
isolates, 14Neisseriaspecies, and 7 Moraxellaspecieswere tested with similar
techniques. Nopositive reactionswereobserved. A costeffectivenessstudy using
5, 10, and 20 ulofthegonococcalreagent was undertakentoreduce thecostof
the test. When 10 and 20
,ul
ofreagent wereused,no difficultywas encounteredininterpretingthereaction. The coagglutination techniquewasdifficulttoread
when 5
[lI
of reagent wasused.The numberofgonococcal infections makesit
increasingly important
to evaluate newrapid
methods for the
identification,
treatment, andcontrol of Neisseria
gonorrhoeae
infections.There are three generally
accepted
methodscurrently employed
in laboratories foridentifi-cation of Neisseria
species:
the directfluores-cent-antibody
technique,
carbohydratedegra-dation, and the
staphylococcal
coagglutination
(COA) technique.
Cross-reactivity
has been aproblem with the direct
fluorescent-antibody
technique in the past (3). Different
degrees
offluorescence have been showntobeincorrectly
interpreted,andcertain
fluorescent-antibody
re-agents havenot
always
beenspecific
(3).Recentimprovements have been made (10). Accurate
andspecificmethods forcarbohydrate
degrada-tion have been studied for years. Many
tech-niques have beenreportedin theliteratureand
marketed commercially with agar plates, agar
slants,andsemisolid andliquidmedia(2, 4, 6, 8,
15, 16). Erroneous fermentation patterns have
resulted because ofimpure cultures,insufficient
inocula, incorrect composition of maintenance
media, and lackof certain essential growth
fac-tors (5,16). In recent years a rapid slide
coagglu-tinationtestfirst introduced byKornvall (4, 9)
and mostrecently marketed by Pharmacia
Di-agnostics, Piscataway, N.J. (Phadebact
Gono-coccusTest), has beendevelopedas an
alterna-tive to traditional procedures for confirmatory
identification of N. gonorrhoeae. This COA
methodhas beenshown to besensitive,specific,
andsimple(1, 7, 11, 12).
Thepurpose of this study was to compare the
rapid fermentation method (RFM), which is a
carbohydrate degradation method, with the
Phadebact GonococcusTest.
MATERIALS AND METHODS
Bacterial cultures. Of the 147isolates tested in thisstudy,114werefresh clinical isolates from Walter
Reed ArmyMedicalCenter,7were fromthe Walter
Reed Army Medical Center Reference Strain Collec-tion, 2wereproficiencysurvey strainsobtained from
aCollege ofAmericanPathologysurvey, and 24 were
from the Walter Reed Army Institute of Research
Collection (15 stock strains and 9 carrier strains). Sources of these organisms are shown in Table 1.
Isolated colonies were presumptively identified by
Gramstain(Scientific Products, Columbia, Md.), mor-89
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90 FUTROVSKY, GAYDOS, AND KEISER
TABLE 1. Sources oforganisms
No. of isolatesof:
Source oforganism N.gonor- N.meningiti- Neisseria Moraxella rhoeae dis species" species
Clinical isolates
Cervix 37 1
Vagina 14
Urethra 32
Throat 1 11 2
Blood 3 2
Rectum 3
Sputum 1 1
Cerebrospinalfluid 2
Prostaticmassage 1
Penis 1
Intrauterine device 1
Skinlesion 1
Stock cultures
WRAIRa Collection(sourcesunknown) 24
Proficiencysurvey 1 1
WRAMCh Reference Strain Collection 1 3 3
aWRAIR, Walter ReedArmy Insititute of Research.
bWRAMC, Walter Reed Army Medical Center.
'Sucrose-, maltose-, and glucose-positive Neisseria species, with the exception of one sucrose-negative,
lactose-,glucose-,andmaltose-positiveN.lactamica culturein theWRAMCReferenceStrainCollection.
phology, and oxidase(PathoTec; General Diagnostics, Morris Plains, N.J.) reaction. All Neisseria isolates
were characterized by theRFM.Final identification oforganisms otherthanNeisseria wasperformed by conventional biochemical methods(17).
All strainswereinitially cultured onboth Martin-Lewisagar(BBLMicrobiology Systems,Cockeysville, Md.) and chocolate agar (BBL) andincubated
over-nightat35°C ina5to8%CO2incubator. After18to 24h ofgrowth, organismswere subculturedon choc-olateagarforpurificationbeforetestingwith the RFM and theCOAtechnique.
Preservation and storage of organisms. All
clinical isolateswerefrozenat-25°C in1-mlaliquots
of10%skimmilk(DifcoLaboratories, Detroit, Mich.) afterbeing testedbytheRFM and theCOAtechnique.
Strains were frozen for a maximum of 2 months, thawed, subculturedonchocolateagar, incubated for 18h at35°Cina5to8%CO2incubator,and retested
with theCOAtechnique.
COA test. The Phadebact Gonococcus Test is a coagglutination procedure consistingoftwo reconsti-tuted reagents: agonococcal reagentcontaining spe-cific immunoglobulin G anti-gonococcal antibodies
produced inimmunizedrabbits andcoupledto heat-killedstaphylococciby protein A and a control reagent
containingimmunoglobulinGproducedin nonimmu-nized rabbitsandcoupledtothestaphylococci. Both reagentswerereconstitutedaccordingtopackage di-rections with 1.5 ml of buffersolution, transferredto labeledtubes,andcentrifugedfor10minat1,500xg. Thesupernatant was decanted, and 1.5ml ofbuffer was addedto resuspendthe button. Thesuspension
was then transferred to the original vials and was
ready foruse.The twoproceduresrecommended for
carrying out the test were followed. In the direct procedure, 1 drop each of both the gonococcal and
controlreagents wasplacedon apetridish (150 by 15 mm) anddivided by a waxpencil into sections, with
the colonies being tested emulsified in each reagent.
After mixingwasdone with a wooden applicator stick, thepetri dish was rocked gently for 1 to 2min and observed for coagglutination. Strength of the
co-agglutination reaction was graded from 0 to 3+,
with 0 indicating no reaction and 3+ indicating a strong reaction.Positive reactions showed3+ aggluti-nation in thegonococcalreagent, with thecontrol rea-gent showing 0 or 1+ agglutination. A positive
re-action could also be shown with 2+ agglutination in thegonococcalreagentbut with only0agglutination
inthe controlreagent. In the alternateprocedure, a
suspension ofthe organismequal to at least a no. 9
McFarland turbiditystandard was mixed in 1 ml of 0.9% sodium chloride. This saline suspension was
boiledfor 20min,and 1drop ofit was used to carry outthetest.
RFM. The Brown modification (2) of the original RFMofKellogg and Turner (8) was further modified for use in ourlaboratory. The changes included: (i)
increasing the phenol red to 0.3 ml per 100 ml of
buffer-salt solution; (ii) premixingthe buffer-salt
so-lution with filter-sterilized (0.2-ytm Nalgene filters; Nalge Co.,Rochester, N.Y.) 10% solutions ofglucose, maltose, sucrose, and lactose (Difco); (iii) dispensing
0.3-mlaliquotsintocapped plastictubes(12by75mm;
ScientificProducts, Columbia, Md.);and(iv)freezing aliquotsfor up to 6 monthsat-20°C.
Abetterhomogeneous suspension was obtainedby usingasterile cotton swab(Hardwood ProductsCo., Guilford, Maine)rather than the recommended 3-mm-J. CLIN. MICROBIOL.
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diameterwireloopfor inoculation. Documented stock
culturesof N.gonorrhoeaeand Neisseria meningiti-dis wereused ascontrols in each test. Each set of
carbohydrate tubeswasheavily inoculated (with an
amount equaltoatleasta no.9McFarlandturbidity standard) with unknown or control organisms and incubatedina35°C drybath foramaximum of4h. Negative reactionswere either pink or orange-pink, andpositive reactionswereyellow.
RESULTS
To check the specificity and sensitivity of the
COA method as compared with the RFM, 114
clinical isolates and 33 laboratory strains of N.
gonorrhoeae and other bacteriawereexamined
(Table 1).
Testing with thedirectCOAmethodwasdone
on 46 N. gonorrhoeae and 2 N. meningitidis
cultures. Only 28 (61%) of the N.gonorrhoeae
cultureswere correctly identified by the direct
COA method. The 2 N. meningitidis cultures
wereshowntobe negative. The remaining18N.
gonorrhoeaecultureswereconsideredtobe
non-interpretable duetoanonspecific reaction in the
controlreagent. The 48 cultureswere retested
with the alternate method of boiling. All 46 N.
gonorrhoeaeculturesgaveclearpositive results,
and the 2 N. meningitidis cultures gave clear
negative results. We therefore decidedtoboilall
cultures whenusing the COA method.
The results of the COA method and the RFM
are shown in Table 2. The two isolates of N.
gonorrhoeaethatwerenoninterpretable bythe
COAmethodhad 3+coagglutinationin the
gon-ococcal reagentwith 2+ coagglutination in the
control reagent. Both cultureswere rechecked
for purity and found to be pure. They were
reboiled, and the same coagglutination results
were demonstrated. These twoorganisms were
determinedtobeN.gonorrhoeae bythe RFM.
Two otherisolates of N.gonorrhoeae incorrectly
identifiedasN.meningitidis bythe RFMwere
shown to be N. gonorrhoeae by the COA
method. Thesestrainsweresent tothe Centers
for Disease Control, Atlanta, Ga.,where
exten-sive evaluation by the direct
fluorescent-anti-body technique and carbohydrate degradation
confirmed themasN. gonorrhoeae. Itwaslater
determined that some of the maltose powder
used for testing these two strains had
deterio-ratedtoglucose. WVhen the isolateswereretested
withnewmaltose, bothwerecorrectly identified
asN. gonorrhoeae.
The decision matrix (13) of the Phadebact Gonococcus Test (Table 3) shows the sensitivity
to be 97.8%, with a specificity of 100%. The
positive predictive valuewas100%, and the
neg-ative predictive valuewas96.4%.
DISCUSSION
The results obtained show the Phadebact
Gonococcus Testtobeasreliableasthe RFM.
The 100% specificity shows that therewere no
false-positives with the COA method. The 100%
positive predictive valuewe obtained indicated
thatapositivetestwith theCOA methodmeant
that the strainwasN.gonorrhoeae. A negative
result by the COA method will beaccurate96.4%
ofthe time, with 3.6% false-negatives. Because
of these false-negatives in the COA method,
oxidase-positive, gram-negative diplococci
mor-phologically resembling N. gonorrhoeae should
betested by another method. For small
labora-tories withoutabackupsystem, the Phadebact
Gonococcus Test should be sufficient if
nonin-terpretable or suspicious unconfirmed isolates
are sent to a reference lab for identification.
Recently, several oropharyngeal isolates of
Neis-seria lactamica were reported to yield
false-positive results with the Phadebacttest (1). We
observednocross-reactivityfor the11Neisseria
species throat specimenswetested(Table 1). In
addition,nocross-reactivitywasobserved in the
oneN. lactamicaisolate from theWalter Reed
Army Medical Center Reference Strain
Collec-tion(Table 1). Until further data is obtainedwe
would recommend that at least all
oropharyn-geal specimens yielding positive coagglutination
results should havean
o-nitrophenyl-,8-D-galac-TABLE 2. COA method and RFM results with147clinical and stockorganisms
No. of No. positive No. negative noninterpretable Organism strains
tested Phade- RFM Phade- RFM Phade- RFM
bact bact bact
N.gonorrhoeae 93 91 91 0 2a 2 0
N.meningitidis 33 0 33 33 0 0 0
Neisseriaspecies 14 0 14 14 0 0 0
Moraxella
species'
7 0 7 0aIncorrectly identifiedbytheRFMasN.meningitidis.
b The RFMcannotbe usedtoidentifyMoraxellaspecies (17).
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92 FUTROVSKY, GAYDOS, AND KEISER
TABLE 3. Decision
matrixa
of the Phadebact Gonococcus TestOrganism No.positive No. negative
N.gonorrhoeae 91 2
N.meningitidis
Neisseriaspecies 0 54
Moraxellaspecies
a For areview,seereference 13.
topyranoside(ONPG)testruninconjunctionto
eliminatefalse-positives.The need foranONPG
testonallpositive Phadebact specimens
regard-less ofsourceofisolation is unconfirmedatthe
presenttime.
The RFM and the COA method are both
rapid and reliableassays,withtheCOA method
havingsomeadvantages. Both methodsare
sim-ple to operate once a pure culture has been
obtained. TheRFM,however,canonly be used
in a laboratory thathas facilities available for
reagent preparation, whereas theCOAmethod
can be used in hospitals, clinics, or doctors'
offices that doordonothave facilitiesavailable
topreparereagents. TheCOAmethod hasa
2-month expiration date when the reagents are
reconstituted and stored at 4°C, whereas the
RFMreagentsarestableforupto6months but
mustbekeptat-20°C orbelow. Confirmatory
identification of Neisseria species can be
ob-tainedwithin 4 hbytheRFM,butconfirnatory
identification of N. gonorrhoeae is obtainable
within1hwiththe COA method.Studiesusing
5-, 10-, and20-t,lamountsofPhadebactreagents
wereperformedon30N.gonorrhoeae cultures.
The 10- and20-,ulamountsproduced
coagglutin-ationclearlydiscernible with the nakedeye,but
the
5-u1l
tests were difficult to interpretunlessreadunderamicroscope. Therefore,the reagent
costof$1.25perPhadebact test couldprobably
be reducedsomewhatbytheuseoflessreagent.
Finally, it wasdeterminedfrom thisstudythat
the alternate method ofboilingshould be used
routinelyasopposedtothedirect method.
Boil-ing isnecessary toeliminateautoagglutination.
This finding also suggests that the Phadebact
antibodyis directed againstheat-stable,tightly
boundgonococcalcellsurfaceantigen(s).Boiling
mayreservethecommonantigen(s) which
nor-mal rabbit sera antibody is directed against,
exposing the most specific gonococcal surface
antigen(s),thusincreasing antigenicity.
Contrary tothe Phadebact Gonococcus Test
packageinsert(14),wefoundthatisolates could
befrozen in 10% skim milk foramaximum of 2
months, thawed, subcultured, andretyped. All
147
specimens
werefrozenin 1mlof skim milkafterinitialtesting bytheCOA methodand
.the
RFM.All specimenswerefrozenat-25°Cfor1
to2months,thawed, plated onchocolateagar,
checkedfor purity, and retested for
coagglutin-ation. Again, 91 of93 N.gonorrhoeae cultures
were positive and 2 werenoninterpretable, and
the other 54 isolates were clearly negative. In
conclusion, the Phadebact Gonococcus Test
uti-lizing boiling has been found to be arapidand
accurate techniquehaving thecapabilityto
iden-tify N. gonorrhoeae from clinicalsources.
ACKNOWLEDGMENTS
We thank W. J. Brown, Centers for DiseaseControl, At-lanta, Ga., for helpfulsuggestions in modifying the RFM for usein ourlaboratory and Joel S. Lewis, Centers forDisease Control,forhelpinconfirming two strains of N. gonorrhoeae.
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