0095-1137/82/070077-05$02.00/0
Sensitivity,
Specificity, and Predictive Values of the Limulus
Lysate Assay for Detection or Exclusion of
Gonococcal
Cervicitis
VINCENTA. SPAGNA,1'2RICHARD B.PRIOR,l*ANDGEORGEA. SAWAYA3
Division ofInfectious Diseases, Department ofMedicine,1 and Department of Obstetrics and Gynecology,3 The Ohio State University College of Medicine, Columbus, Ohio 43210, and Venereal Disease Clinic, City
Departmentof Health, Columbus, Ohio432152
Received4February1982/Accepted 2 April 1982
TheLimulus amoebocyte lysate (LAL) assay was evaluated for its ability to
detect or exclude gonococcal cervicitis in two groups of women. The first
(positive) group consisted of 100 untreated women who were referred to the
venereal disease clinic with culture-proven gonococcal cervicitis. The second
(negative)group consisted of 50 normal volunteers who were evaluated on two separate occasions. In the firstgroup, Gram stains and repeat cervical cultures
were 53 and93% sensitive, respectively. Inthe second group, Gram stains and
cultures were negative. For the LAL assay, ectocervical mucus was removed
witha sponge,and adepyrogenated cotton-tipped swab was thenused to collect endocervicalspecimens. The swabwasplaced in1mlofdiluent(1:1dilution), and
serial twofold dilutions weremade and testedfor endotoxin by the LAL assay.
Incubation was carried out at37°C for 30 min; positiveor negative resultswere
indicated by gelation or lack of gelation, respectively. At a dilution of 1:256,
sensitivity and specificity of theLALassay were57 and 99%,respectively. The positive predictive values ranged from 36.5 to 97.4% for theoretical prevalence
ratesof1 to40%.Atadilution of 1:8, the sensitivity and specificitywere 100 and 78%, respectively. At this dilution, the negative predictive value was 100%
regardlessof the prevalencerate.Thus, these preliminaryresults showthat at the
higher dilution, the LAL assay was comparable to Gram stain in diagnostic
accuracy ofgonococcal cervicitis, and if used as a screening test at the lower
dilution, a negative LAL assay would exclude women without gonococcal
cervicitis.
Despite existing control measures, gonorrhea
remainsepidemicwith anestimated world
inci-dence of approximately 70,000,000 (16). Over
1,000,000 cases are reported annually in the
United States(4), and it is estimated that fourto
five times this number are seen in the private
sectorbypracticing physiciansandnotreported
(1,7, 13). Although the factors which contribute
to the high prevalence ofgonorrhea are varied
and complex, the asymptomatic carrier is the
mostimportantelement in disease transmission.
Unsuspected gonococcal infection occurs in
bothmales andfemales; however, women
con-stitute the greatest reservoir of asymptomatic
disease and offerthe best means of
identifying
asymptomatic men(10, 16).
Despite efforts to develop methods for an
immediate diagnosis of gonococcal
cervicitis,
the Gramstain remainstheonlyaccepted
proce-dure in clinical medicine (22). Examination of
Gram-stained smears of cervical exudate
by
experienced personnel detects between 50 and
65% ofpatientswhosubsequentlyhavepositive
cultures for Neisseriagonorrhoeae (2, 3, 5).
We have previously reported ourexperience
with the Limulus amoebocyte lysate (LAL)
as-sayin therapid, presumptivediagnosisof
gono-coccal urethritis (15, 17) and have used it to
evaluategonococcal cervicitis (18).However,in
thecervicitis study,welimitedourpatient
popu-lationto women withpurulentendocervical
se-cretions and usedacumbersomepipettemethod
witharubber suction bulbtocollectspecimens.
Wereport here ourresultswith the LAL assay
forevaluating(i) gonococcalcervicitisinwomen
with both purulent and nonpurulent
endocervi-cal exudates and(ii)normalwomenbyutilizinga simple collection procedure and a 30-min
incu-bation period. From these results, the positive
andnegative predictivevaluesofthe LALassay
werecomputed.
(This workwaspresented in part atthe First
SexuallyTransmitted Diseases WorldCongress,
SanJuan,PuertoRico,15to21November, 1981.)
77
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78 PRIOR, AND SAWAYA
MATERIALS ANDMETHODS
Patient population. One-hundred women with
un-treated culture-proven gonococcalcervicitis whowere
referredtothe Columbus HealthDepartmentVenereal
Disease Clinic formed one study group. The mean
duration between initial culture and repeat
examina-tionwas11.2days,witharangefrom 2to28days.The meanageof the patientswas22.0years,witharange
from 18to36years.
The othergroupconsisted of 50 normal adult female
volunteers whowereevaluatedontwoseparate
occa-sions by one of the authors (G.A.S.) in a private practice facility. Thenormal volunteerswerewithout
signs orsymptoms ofgenitourinary diseaseandwere monogamousforatleast thepreceding12months. The mean age of the volunteers was 27.3 years, with a
rangefrom 20to43years.
Patients wereexcluded from eithergroupifthey(i)
were under 18yearsofage ormenopausal, (ii) were
pregnant,(iii) lackedacervixorhadsurgical injuryto the cervix, (iv) were menstruating at the time of
examination, (v)admittedtoantibioticusagewithin10
days of examination, or (vi) failed to give informed
writtenconsent.
Diagnostic procedures. Each patient and volunteer
underwent a standardized interview concerning:
de-mography, sexual and venereal disease history, and presentillness; examinationof thegenitaliaand ingui-nal lymph nodes; and a pelvic examination which
included theuseofaspeculumtovisualize the cervix.
Metalorplastic speculawereused and lubricated with water. Both ecto- and endocervical samples were
collectedby using cotton-tippedswabs(Chaston,
Mel-ville, N.Y.) whichweredepyrogenated by heating at
210°C for 30 min (21). The ectocervical sample was collected firstby wipingthe surface of the cervix with aswab. The swabtipwasthen broken off inaplastic tube containing1 ml ofpyrogen-free water,and this constituteda1:1 dilution for thesubsequent quantita-tive studies of endotoxincontent. Aftercollection of
the ectocervical sample, the cervical surface mucus wasremoved by wiping the cervixwithapyrogen-free foamspongePrep-Swab (Truly Magic Products, Inc.,
Buffalo, N.Y.) which was trimmed so that the end surface measured approximately 1.5 by 1.5cm. The endocervical samplewasthen collectedby insertinga
cotton-tipped swab into the endocervical canal
ap-proximately 1.5 cm for 10 s. Care was takennot to touch the vaginal vault with the swab during the collection procedure. The swab tip containing the
endocervical sample wasalso broken off in aplastic testtubecontaining 1 mlof pyrogen-freewater.
For culture, sterile cotton swabs were inserted
approximately 1 cm into the endocervical canal and
blindly into the anal canal; samples were streaked
directly ontoThayer-Martin medium for isolation of N.gonorrhoeae. The plateswereincubated for48hat
350C in 5%CO2. Neither additionalbacterial cultures norcultures for virusesorchlamydiaweredone.
Thesameprocedurewasusedtoobtainan
endocer-vical specimen for Gram staining and subsequent
microscopicexamination.
Laboratorymethods.Gram-stainedsmearsof cervi-cal exudate were examined under
1,000x-magnifica-tion oil immersion forgram-negative diplococci and
polymorphonuclear cells. Smears were considered
positive iftypical gram-negative diplococciwere locat-ed intracellularly in polymorphonuclear cells. All iso-lates grown on theThayer-Martin medium were
con-firmed as N. gonorrhoeae by typical sugar fermentation reactions. In the normal volunteer popu-lation, absence of gonococcal cervicitis required that thecervical Gram stain smears and cultures takenon
bothoccasions were negative for N. gonorrhoeae. LAL assay. Theendotoxincontentin both the
ecto-and endocervical samples was quantitatively deter-minedwith the LAL assay. The cervicalsampleswere
Vortex mixed, and 0.1 ml was transferred toatube containing 0.7 mlofpyrogen-freewater(1:8dilution). Serial twofold dilutions were then made in water, changing pipettes between each dilution, to a final dilution of 1:1,024. After dilution, 0.25 ml of each dilution was addedtosingle-testPyrogent vials (Mal-linckrodt, Inc., St. Louis, Mo.), mixed, incubated undisturbed at 37°C inaheatingblock for 30 min, and read. A firm gel that remained adherenttothebottom of the vial whencarefullyinvertedwasinterpretedas a
positive test; the absence offirmgelation was inter-pretedas anegativetest.Theminimumsensitivity of the LAL assaywasthedetection of0.3 ngof Esche-richia coliendotoxin standard per ml, lot EC-2 (Bu-reau ofBiologics, U.S. Food and Drug Administra-tion,Bethesda, Md.).
In addition to the use of water as a diluent, the endotoxincontentin eachsamplewasalsodetermined by using a5% aqueous solution ofametallo-modified polyanionic dispersing agent (Pyrosperse, Mallinck-rodt) as the diluent. After the removal of 0.1 ml from thecervical samples for assay in water, 0.05 ml of the dispersingagentwasaddedtothetubecontainingthe swab. The tubes were Vortexmixed, and 0.5 mlwas
removed andadded to a tube containing 0.5 ml of the 5% dispersing agentasthediluent(1:2dilution); serial twofold dilutions were then made in this diluent to a final dilution of 1:1,024. After dilution, 0.25 ml was removedfrom each tube and added to LAL single test vials which were incubated and read as previously described.
Analysisof data.Results of the LAL assays, Gram-stained smears, cultures, and pertinent clinical find-ings were entered into a Hewlett-Packard model 9825Aprogrammable computer for subsequent deter-mination of correlation. The chi-square method and Student's t test were used for determination of statisti-calsignificance. The positive and negative predictive values were computed by using the methods described by Galen (8).
RESULTS
Clinicalstudies.Thedemographic and clinical
features ofthe 100 patients withuntreated
cul-ture-proven gonococcal cervicitis revealed that
amajority(86%)of patients had sexual relations
with more than one partner during the preceding 12months, and53%had intercourse during the
period between initial culture and subsequent
evaluation and treatment. Sixty percent of the
patients denied symptoms ofgenitourinary
dis-ease before either examination. However, 7%
developed definitesigns and symptoms of pelvic
inflammatory disease (fever, cervical motion
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TABLE 1. Sensitivity of LAL test at various dilutions of cervical samples in two diluents for 100 patients withculture-proven gonococcal cervicitis
Sensitivity'(%)atdilution: Sample Diluent
1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 -1:1,024
Ectocervical Water NDb ND % 93 86 83 74 69 57 51
5% DAC 100 99 97 96 95 91 84 78 70 55
Endocervical Water ND ND 95 92 83 74 60 47 30 17
5% DA 100 100 100 97 92 83 69 57 41 23
0Probability that a patient with gonococcal cervicitis will have a positive test.
bND, Not done.
cDA,Dispersing agent.
tenderness, adnexal tenderness, and masses)
duringthetime betweeninitial culture and
sub-sequent therapy.
Onrepeatexaminationbefore therapy, visual
inspection of the cervix revealed inflammatory
changes (erythema and friability) in 57% and
purulent (yellow or green) endocervical
secre-tions in 60% of the patients. Examination of
Gram-stainedsmearsofendocervical secretions
revealed intracellular gram-negative diplococci
in53% of the patients. Repeat cultures of
cervi-cal specimens grew N. gonorrhoeae in 93% of
thepatients. Ofthesevenpatients with negative
cervical cultures forgonorrheaonrepeat
evalua-tion,positive rectal cultures forN. gonorrhoeae
were obtained in three. Overall, positive rectal
cultureswere obtainedin28% ofthepatients. Clinical evaluation of the 50 normal volun-teersrevealednoevidenceofcervicitisoneither
ofthe two examinations. Gram-stained smears
andculturesforN.gonorrhoeaewere
universal-ly negativein thisgroup.
LALstudies.Thesensitivityof the LALassay
at various dilutions of ecto- and endocervical
samples diluted in water and 5% dispersing
agentfor100womenwith culture-proven
gono-coccalcervicitisisshown in Table 1. Inwater,a maximumsensitivityof 96%wasobtainedatthe
lowest dilution tested (1:8), whereas in the
dis-persing agent, a sensitivity of 100% was
ob-tained forendpcervical samplesdiluted 1:8. The
increase insensitivity by the dispersingagentfor
endocervical samples was statistically
signifi-cant(P < 0.05).
The specificity of the LAL assay at various
dilutions ofecto- and endocervical samples
di-luted in water and 5% dispersing agent for 50
normal volunteers seen on two separate
occa-sions is shown in Table 2. At the breakpoint
dilution giving maximum sensitivity(1:8) (Table
1), specificity wasbetween 66and 67% for the
ectocervical samples in both diluents. However,
forendocervical samples, specificity increased
to 79% in water and to 78% in the dispersing
agent.The differenceinspecificitiesbetween the
two diluents was not significant (P > 0.05).
Maximum specificity (99%) was obtained at a
dilution of 1:256 for theseendocervicalsamples
in the dispersing agent; corresponding
sensitiv-itywas 57% (Table 1). Of the 57 patients with
positive LAL assays at a dilution of1:256, 39
hadpurulentand 18hadnonpurulent
endocervi-calexudates.Therewas nosignificant difference
(P > 0.05) in sensitivity between purulent and
nonpurulent endocervicalsecretionsatthis
dilu-tion.
Predictive values. The predictive values of
positiveandnegativeLALtestresultson
endo-cervical samplesdiluted in thedispersing agent
for various theoreticalprevalenceratesof
gono-coccal cervicitis are shown in Table 3. The
positive predictive values were computed by
TABLE 2. Specificityof LALtestatvarious dilutions of cervical samplesintwodiluentsfor50 normal volunteersseen on twoseparateoccasions
Specificity'(%)atdilution: Sample Diluent
1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 .1:1,024
Ectocervical Water NDb ND 67 68 73 75 77 85 90 90
5%DAC 58 64 66 68 69 72 74 77 84 90
Endocervical Water ND ND 79 82 88 94 99 99 99 99
5%DA 69 74 78 80 81 86 93 99 99 99
0Probability thatapatientwithoutgonococcalcervicitis willhaveanegativetest. bND, Not done.
cDA,Dispersing agent.
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TABLE 3. Predictive values of positive and negative LAL assaysonendocervicalsamples
diluted in5%dispersing agent for various populationsofwomenwith different prevalencerates
ofgonococcal cervicitis
Prevalence of Predictive value Predictive value gonococcal ofapositive ofanegative cervicitis(%) test(M)Y test(%)b
1 36.5 100
5 75.0 100
10 86.4 100
20 93.4 100
40 97.4 100
aProbability that a patient with a positive LAL assay at a 1:256dilution will havegonococcal cervici-tis.
bProbability that a patient with a negative LAL assayat a1:8dilution willnothavegonococcal cervici-tis.
using a sensitivity of57% and a specificity of
99%,whichwere obtainedatthe 1:256 dilution.
The negative predictive values were computed
by usingasensitivity of 100% andaspecificityof
78%, which were obtained at the 1:8 dilution.
Negative predictive values were 100%
regard-lessof the prevalence rate.
DISCUSSION
Our initialexperiencewiththe LAL assayas a
diagnostic adjunct in the evaluation of
gonococ-calcervicitiswasrestrictedto womenwith
copi-ous purulent cervical secretions and involved
cumbersome equipment for sample collection
(18).Althoughperformance of theLALassayin
this selectgroupwasremarkablewitha
sensitiv-ity of94%andaspecificity of100%,asignificant
number of women with gonococcal cervicitis
have no clinical signs of a purulent cervical
exudateorinflammation of the cervical canal.In
thepresentstudy, thenatureof the endocervical
exudatesdidnotsignificantly affect the
sensitiv-ity of the LALassay.Recently,Young et al. (24)
evaluated the LALassay inarandomgroup of
women and collected samples of endocervical exudate with apolythenecapillarytube attached
to a5-mlsyringe. These workersconcluded that
the LAL results correlated with conventional
microbiological evidence of gonorrhea. They
also noted that variation in sample volume as determinedbymeanprotein concentration
prob-ably accounted for the majority of discrepant
LAL results.In the studyreported here,
depyro-genated cotton-tippedswabs wereused to
facili-tate sample collection and to minimize
varia-tions in sample volume. Addition of the
dispersing agent to the water diluent also
im-proved sensitivity significantlybutdidnot affect
specificity. Although the reasons are
specula-tive, the freeing of bound endotoxin and the
dispersion of mucoid samples from the swab
fibers mayprovidean explanation.
The ectocervix was an unsuitable site for
samplecollection sincesamplestaken from
nor-mal volunteers revealed significant endotoxin
contamination. This contamination was
pro-duced by the normal microbial flora of the
genitaltract, which contains a myriad of
gram-positive and gram-negative
(endotoxin-contain-ing) aerobic and anaerobic bacteria(6, 11, 14).
However, thecervix harborsalimitedvarietyof
microbes compared with the vagina, and these
organisms are found in lower numbers at the
cervix (6).Thesefindingssupport the conclusion
ofcervicalbiopsy studies which reveal
coloniza-tion of the endocervix by small numbers of
organisms (19). Therefore, testing endocervical
samples obtained after the removal ofcervical
surfacemucuswithapyrogen-freefoam sponge
produced results which showed a reduction of
endotoxin contamination with a concomitant
increase in specificity ofthe LAL assay.
Fur-thermore, endocervical samples taken from
women with gonococcal cervicitis contained
largequantities ofendotoxinproduced by
gono-cocci, which have been shown to colonize the
endocervix in average numbers of 105
colony-forming units (12).
The cultural yield of N. gonorrhoeae on
re-peat cervical and rectal sampling (96%)
corre-sponded with results noted by other
investiga-tors who reported that 95 to 98% of infected
women wereculture-positiveon asingle
evalua-tion inaspecialty clinic with adjacent laboratory
facilities (2, 20, 23). Although culturalrecovery
ofN. gonorrhoeae was accomplished within 2
days, the additional time expended in notifying
patients and awaiting their subsequent
presenta-tion for treatment resulted in an average delay
period of11days. As a consequence,53% of the
patients had sexual intercourse, and 7%
devel-oped signs and symptoms ofpelvic
inflamma-torydisease before treatment. Consideringthat
approximately 200,000cases ofgonococcal
pel-vic inflammatory disease occur annually in the United States and that this clinical entity is a
relatively early complication of gonorrhea (9),
theimportanceof rapiddiagnostic tests is
appar-ent.
Inevaluation ofwomen forgonococcal
cervi-citis, the LAL assay when performed on
endo-cervical samples diluted 1:256 in the dispersing agentcomparedfavorablywith the Gram stain in
diagnosticaccuracy. Thesensitivities and
speci-ficities were57%and99%, respectively,forthe
LALassay and53%and100%,respectively, for the Gram stain. However, Gram stain
interpre-tation of cervical specimens requires the
exper-tise of trained microscopists, and, as a
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quence, most facilities rely solely on cultural recovery to diagnose gonococcal cervicitis. If
trained microscopists are unavailable for Gram
staininterpretation, use of the LAL assay may
be appropriate since (i) it is easy toperform by
those unskilled in laboratory procedures, (ii)
results are available within 30 min of sample
collection, (iii) the presence or absence of an
adherentgelasindicators ofa positive or
nega-tive testallowsforobjective standards inresult
interpretation, and (iv)the sensitivity of 57% is
within the usualrangeofsensitivity (50 to 65%)
determined by Gram stain (2, 3, 5). However,
the prevalence of the disease in the population
being testedmustbeconsidered in interpretinga
positive test since the positive predictive value
varieswith the prevalence of the disease(Table
3).
The absenceofspecific signsandsymptomsin
women infected with gonorrhea has prompted
mass screening programs, using cervical
cul-tures. In the United States during 1979, there
were8,778,406screening cultures, with 392,283
(4.5%) positive for N. gonorrhoeae (4). The
economic burden imposed by this approach is
apparent with the large number of negative
cultures. Thenegative predictivevalueofatest
is theprobabilitythatapersonis free of disease
if the test result is negative. In evaluation of
uninfectedvolunteers,anegativeLAL assay on
endocervicalsamples diluted 1:8in the dispers-ing agent occurred in
78%;
all of the patientswith known gonococcal cervicitis had positive
LAL test results at this dilution. Thus, the
predictive value of a negative LAL assay to
exclude women without gonococcal cervicitis
was 100%. Since sensitivity of the LAL assay
was 100o at this lower dilution, prevalence of
thedisease hadnoaffectonthenegative
predic-tive value(Table 3).
Thesepreliminaryresults with theLAL assay
intwowell-definedgroups ofwomenwill
hope-fullyestablishguidelines for further research of
thistestinevaluation ofwomenforgonococcal
cervicitis.
ACKNOWLEDGMENTS
WethankJosephG. Lossickand his staff of the Columbus HealthDepartmentVenereal Disease Clinic and the nursing staff inG.A.S.'s officefortheircooperationand assistance.
Wealso thank the manywomen whovolunteeredto
partici-patein thisstudy.
Thisstudywassupportedin partbyagrant from Mallinck-rodt, Inc.,St.Louis,Mo.
LfIERATURECITED
1. Barlow,D.1979.Sexuallytransmitteddiseases,thefacts,
p. 19-29.OxfordUniversityPress,NewYork. 2. Barlow,D.,K.Nayyar,I.Phillips,andJ.Barrow.1976.
Diagnosis ofgonorrhoea in women. Br. J. Vener. Dis. 52:326-328.
3. Barlow, D., andI. Phillips. 1978. Gonorrhoeae in wom-en-diagnostic, clinical and laboratory aspects. Lancet i:761-764.
4. Centers for Disease Control. 1979. Sexuallytransmitted disease (STD) statistical letter, issue no. 129, p. 9-40. Centers for DiseaseControl, Atlanta,Ga.
5. Chipperfield,E.J.,andR. D. Catterall.1976.Reappraisal ofgram-stainingand culturaltechniquesfor thediagnosis ofgonorrhoeainwomen.Br.J. Vener. Dis. 52:36-39. 6.Corbishley, C.M.1977.Microbial flora of thevagina and
cervix. J. Clin. Pathol. 30:745-748.
7. Fleming,W.L.,W.J. Brown, J.F.Donohue, andP. W.
Branigin.1970.Nationalsurveyof venereal disease
treat-edby physiciansin 1968. J.Am.Med. Assoc.
211:1827-1830.
8. Galen,R.S.1980.Statistics,p. 41-68.In A. C. Sonnen-wirth and L.Jarett(ed.),Gradwohl'sclinical laboratory methods anddiagnosis, 8th ed. C. V. Mosby Co., St. Louis,Mo.
9. Hager,W.D.,and P.J.Wiesner.1977.Selected
epidemi-ologic aspectsofacute salpingitis:areview. J. Reprod. Med. 19:47-50.
10. Handsfield, H. H., T. 0. Lipman, J.P. Harnisch, E.
Tronca,and K. K. Holmes. 1974. Asymptomatic gonor-rhea inmen.N.EngI.J.Med. 290:117-123.
11. Levison, M. E.,L.C.Corman, E.R.Carrington,and D.
Kaye. 1977.Quantitativemicroflora of thevagina.Am. J.
Obstet.Gynecol. 127:80-85.
12. Lowe,T.L.,andS.J. Kraus. 1976.Quantitationof
Neis-seriagonorrhoeaefromwomenwithgonorrhea.J.Infect. Dis. 133:621-626.
13. Morton, R.S. 1977. Gonorrhoea, p. 204-234. W. B. Saunders Co. Ltd.,Philadelphia.
14. Onderdonk,A.B.,B. F.Polk,N. E.Moon, B. Goren,and
J.G.Bartlett. 1977.Methods forquantitative vaginalflora studies. Am. J. Obstet. Gynecol.128:777-781.
15. Prior, R.B., and V. A. Spagna. 1981. Application ofa
Limulustestdevice inrapidevaluation ofgonococcaland nongonococcal urethritis in males. J. Clin. Microbiol. 14:256-260.
16. Rein, M. F. 1977.Epidemiology of gonococcal infections, p. 2-17. In R. R. Roberts (ed.), The gonococcus. John WileyandSons,NewYork.
17. Spagna, V.A., R. B. Prior, and R. L. Perkins. 1979. Rapid presumptive diagnosisofgonococcal urethritis in
menbythelimuluslysatetest.Br. J. Vener. Dis. 55:179-182.
18. Spagna, V.A., R.B. Prior, and R. L. Perkins. 1980.
Rapid presumptive diagnosisofgonococcalcervicitisby
the limulus lysate assay. Am. J. Obstet. Gynecol. 137:595-599.
19. Sparks, R.A., B.G.A. Purrier, P.J. Watt, and M.
Elstein. 1977. Thebacteriology of the cervical canal in relationtotheuseofanintrauterinecontraceptive device,
p.271. In V. Insler andG. Brettendorf(ed.),The uterine cervix inreproduction. GeorgThiemeVerlag,Stuttgart.
20. Thin,R.N.,andE.J.Shaw. 1979.Diagnosisof gonorr-hoea inwomen.Br. J. Vener.Dis. 55:10-13.
21.Tsuji,K., and S.J.Harrison. 1979. Limulusamebocyte lysate-ameans to monitor inactivation of lipopolysac-charide,p.367-378.InE. Cohen(ed.),Biomedical
appli-cations of the horseshoecrab(limulidae). AlanR.Liss, NewYork.
22. Young, H.1981. Advances in routinelaboratory proce-dures for thediagnosisofgonorrhoea,p.59. In J. R. W.
Harris (ed.), Recent advances in sexually transmitted diseases,no.2.ChurchillLivingstone,Edinburgh. 23.Young, H., A.B. Harris, D. Urquhart, and D. H. H.
Robertson.1979.Screeningbyculture for the detection of
gonorrhoeainwomen. Scott. Med. J. 24:302-306.
24. Young,H.,S. K.Sarafian,and A.McMillan. 1981.
Reac-tivity of the limulus lysate assay with uterine cervical secretions-apreliminaryevaluation. Br.J. Vener.Dis. 57:200-203.
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