Copyright ( 1978 AnmericanSocietyforMicrobiology PrintedVol.in7,U.No.S. A.1
JAMES H. JORGENSEN* ANDJEAN C. LEE
Departments ofPathologyandMicrobiology, The UniversityofTexas Health Science Center atSan Antonio and the Bexar CountyHospitals,SanAntonio,Texas 78284
Received for publication 29 July 1977
The Limulus amoebocyte lysate endotoxin assay wasevaluated as a method
for rapid diagnosis of acute bacterial meningitis ina series of 305patients. The
results of Limulus assays on cerebrospinal fluid (CSF) samples from these
patients were compared with the results for each patient of routine bacterial
cultures and Gram stains. Positive Limulus tests were obtained oninitial CSF
specimensfrom 84%ofpatientswithculture-provenbacterial meningitis,
includ-ing all patients with meningitis due to gram-negative organisms. Initial
Gram-stained smears revealed the presence oforganisms in 68% of the patients. One
patient with pneumococcal meningitis had a weakly positive Limulus assay,
whereas patients with meningitis due to other gram-positive organisms, those
tests.The Limulus assay also demonstratedthepersistence ofendotoxin in the
CSF ofcertain patientsduring antibiotictherapy, especially patientswith
Hae-mophilus influenzae meningitis.TheLimulustestproved tobearapid, reliable
indicator of the presence ofgram-negative organisms in the CSF of patients
suspected of acute bacterialmeningitis.
The mortalityassociated withacute bacterial meningitiscanbegreatly reducedifappropriate antibacterial
therapyisinitiated promptly (17).
The usuallaboratory diagnosticmethodsto
dif-ferentiate bacterial from viral or other agents
ofmeningitisareaculture ofcerebrospinalfluid (CSF) for bacterial pathogens, direct micro-scopic examination ofCSFforbacteriaand leu-kocytes, and measurement ofCSFglucose and
protein concentrations. If treatment is to be
successful, antibacterial therapy must be
initi-ated before culture results become available
(17). However, results ofmicroscopical,
chemi-cal,andhematological examinationsof CSF may beinconclusiveortotally misleading, especially in early bacterial meningitis (7). The
examina-tion ofGram-stainedsmearsofCSF for bacteria
has been shown to yield reliable information
regarding theetiologicalagent involved in only
60 to80% of cases, even in the mostexperienced
hands (2, 25). Therefore, more precise, rapid
methods for the early detection ofmeningitis
Current rapid methods for the diagnosis of bacterial meningitis are mainly immunological techniques for the demonstration of bacterial antigens in CSF.
Counter-immunoelectropho-resis (CIE) has been used for the detection of
meningococcal, Haemophilus, and pneumococ-calantigensinthe CSF ofpatients with
menin-gitis (3,5,10,11, 18, 20). Latex slideagglutination
techniquesfor the detection of these
(22, 26)also appear promising. However, both
antisera that may bedifficult to obtain
commer-cially.The determination of the CSF lactate
concentration (8) may alsohelp to
bacterial from viralorfungal
The Limulus in vitro endotoxin assay has
been usedpreviously for thedetection of
endo-toxin inblood (6, 15, 24) and urine (12, 13) and
as a method for pyrogen testing of
pharmaceutical products (16, 19). This test is currently the most sensitive method available
for the detection of endotoxin. The results of
assay for the rapid detection ofgram-negative
bacteria in CSF appear very encouraging (1,21,
usingthistechnique for the rapid diagnosis of
MATERIALS AND METHODS
Patient group.Adult andpediatricpatients ofthe BexarCountyHospitals,onwhom alumbarpuncture
included in this study.On each CSFspecimen,aGram
stain and culture, as well as Limulus assay, were performed.
Performance ofLimulus assays. CSF samples
forLimulustestingwerecollected insterile, pyrogen-12
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DIAGNOSIS OF BACTERIAL MENINGITIS
freeplasticorglasstesttubes.Two different Limulus
amoebocyte lysate preparations were used in this
study,dependinguponavailability.Amoebocytelysate waspreparedinourlaboratoryby methods previously
described (14),and acommerciallysate preparation, Difco Pyrotest (generously suppliedforthis study by
Difco Laboratories, Detroit, Mich.), was also used.
Thesensitivities of individuallysate preparationswere
determined as previously described (14) by testing with Escherichia coli 0124:B4 lipopolysaccharide, also supplied by Difco Laboratories. Limulus lysate
prepared in our laboratory could detect as little as
Toperform aLimulustest on aCSF specimen with
ourlysate,0.1 mlof the CSFspecimenwasaddedto asterile, pyrogen-free glasstesttube(10 by 75mm) to which 0.1 ml ofLimuluslysate had been added. Forspecimensexamined usingPyrotest,a0.2-ml
sam-pleof CSFwasaddeddirectlytothelyophilized Lim-ulus amoebocyte lysate in a single test vial. Both
positive andnegativecontrolswererunwith eachset ofassays.Thepositive controlconsisted of the
addi-tion of an appropriate volume of a 1-ng/ml stock
tube of theparticularlysatepreparation. Additionof
pyrogen-free salineorwater toanothertube of lysate
constituted a negative control. All Limulus assays wereincubatedat37°C for70mininabacteriological
incubator. Afterincubation, eachtubewasexamined
for the presence of a gel or a definite increase in
viscosity and turbidity compared with the negative
controltube (14).Bothastronglypositive (4+)
reac-tion in thepositive controltubeandanegative
(water-like) reaction in the negative control tube were
Occasionally, the volume of the CSFspecimenwas insufficienttobe processedinthe abovemanner. In such instances,aportionofsterile,pyrogen-freewater (Travenol Laboratories,MortonGrove,Ill.)wasadded
tothe existingamountof CSFtoyieldafinal volume
of at least 0.1 ml. The diluted specimen was then
assayedin themanner described above, exceptthat anadditional0.1mlof waterwasaddedtothePyrotest
vialstobringthe finaltestvolumeto0.2 ml.
CSF specimens from a total of 305 patients
by the Limulus test as well as by standard
bacterial culture and Gram stain. Atotal of74
patients had culture-proven acute bacterial
meningitis(Table 1).Theseincluded 61patients
withmeningitisdue togram-negative organisms
and 13patientsfrom whomgram-positive orga-nisms were isolated. Only 1 patienthad
tuber-culous meningitis, whereas the remaining 230
patients eitherdid not havemeningitis orwere
diagnosed as having aseptic meningitis. All of
the initial CSFspecimens fromthe 61 patients
Limulus tests were negative from all but 2
patients whose CSFs were culture-negative,
from the patient with tuberculous meningitis,
and from 12 of 13 patients with gram-positive
bacterial meningitis. The one exception in the
lattergroup was aweaklypositive(1+) reaction
encountered with one CSF sample from a
pa-tient with pneumococcal meningitis. The two
false-pos-itiveLimulus assayshadnot receivedprior
Gram stainswerepositiveonCSFsfrom 72%
meningitisandonly46% (6/13) ofpatientswith
gram-positive meningitis.Gram stainswere
neg-ativeon allCSFs thatwereculturenegativeand on the CSF from the patientwithtuberculous meningitis.
A total of 22 patients were found to have
persistent endotoxin in subsequent CSF
speci-menswhileon antibiotic therapy (Table 2). Of
thesepatients, 13 had Haemophilus influenzae
isolated from their initial CSF specimens.
Among these, cultures of CSFfromrepeat
the Gram stain was only positive in 1 case,
although Limulus assays continued to be
posi-tive in all 13patients.Incontrast,allofthenine
patients with non-Haemophilus gram-negative
meningitis and persistent CSF endotoxin had
positiveCSF cultures. Only 1 of the 9had
posi-TABLE 1. Results of initialCSF examinationson
305patientssuspected of bacterial meningitis
H. influenzae Neisseriameningitidis E.coli
calcoac-eticus var. anitratus
A. calcoaceticus var.
No.ofpa- Limulus Gram tients positive itaiv
30 5 3
1 2 1
1 1 0
1 1 1
1 1 1
4 0 2
6 1a 3
3 0 1
1 0 0
230 2 0
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14 JORGENSEN AND LEE
TABLE2. Patients withpersistentCSF endotoxin
during antibiotic therapy
Limu- Repeat Repeat Organism isolated patientsNo. of lusitivepos- positiveculture staingram
H.influenzae 13 13 1 1
E.coli 3 3 3 1
Neisseria meningi- 1 1 1 0
Klebsiella pneumo- 1 1 1 0
Flavobacterium 1 1 1 0
P.aeruginosa 2 2 2 0
Acinetobacter cal- 1 1 1 0
tive Gram stainfrom the follow-up CSF
Thetime relationship of positive CSF
endo-toxin assays from patients with H. influenzae
meningitiswasalso examined. With CSF
endo-toxin assays performed after the initiation of
therapy in patients withH. influenzae
menin-gitis, positive Limulusassayswerefoundas
fol-lows:at 24h, six of nine patients; at48h, five
ofseven patients; at 72 h, one of one patient;
andat>72h,twoof sixpatients. All specimens
wereculturenegative for H. influenzae. Ascan
patients with sterile repeat CSF specimens to
haveapersistence of endotoxin 24to72hafter
the initiation of antibiotic therapy, with one
patient demonstrating persistent CSF endotoxin
Two specific cases among this group were
studied in greater detail. Table 3 indicates the
laboratory results on patient J.M., a
7-month-oldchild with H.influenzae meningitis who had
persistent CSF endotoxin for at least 9 days
after the initiation of antibiotic therapy,
al-though the CSF culturewasnegative after48h
oftherapy with chloramphenicol. PatientJ.M.
recovered without apparent neurological
Patient R.P. wasa51-year-oldmale who
de-veloped meningitis with Pseudomonas
aerugi-nosaafteraneurosurgical procedure (Table 4).
The initial CSF Limulus assay was strongly
positive. A CSF specimen obtained 48 h after
the initiation of therapy was both sterile and
Limulus test negative. However, subsequent
samples obtained between72h and11daysafter
the initiation of therapy demonstrated the
growth ofafew colonies ofPseudomonas, but
persistently negative Limulus tests. A repeat
CSF specimen obtained on day 11 of therapy
was again strongly Limulus testpositive. The
patient expired shortlyafter the last CSFsample
A rapid, reliable laboratory method for the
diagnosis of bacterial meningitiswould allow a
prompt initiation of antimicrobial
chemother-apy. Rapid immunological methods, such as
CIE, may detect the presence of antigens of
certain of the pathogensmostoftenencountered
reactive with the specifictestbattery of antisera
will be missed by this technique. The Limulus
endotoxin assayisarapid and reliable method
for the demonstration ofavariety of bacterial
antigens in certain body fluids, including CSF.
In the present study, Limulus assays were
easily interpretable and, when positive, usually
could be read afteronly20 to30min of
incuba-tion. The commerciallysatepreparation
(Pyro-test) was found tobesuperior in sensitivity to
parallel, no discrepancies were found between
results obtained using the two lysate
prepara-tions, althoughpositive reactionswere more
def-inite and occurred sooner with the Pyrotest.
False-negative (or -positive) results were not
observed in those instances in which sterile
wa-TABLE 3. Persistenceof CSF endotoxin for9days inspiteofnegativeCSF cultures inpatientJ.M. Day Cultureresult Gramstain Limulus
0 H.influenzae GNRa 4+
2 Nogrowth NBSb 2+
4 Nogrowth NBS 4+
6 Nogrowth NBS 1+
9 Nogrowth NBS 1+
aGNR, Gram-negative rods. bNBS,Nobacteriaseen.
TABLE4. Persistenceof organisms in CSF cultures for11 days inpatientR.P.althoughmostLimulus
Day Culture result Gram stain Limulus as-result say result
0 P.aeruginosa GNRa 4+
2 Nogrowth NBSb Neg.c
3 P.aeruginosa NBS Neg.
6 P.aeruginosa NBS Neg.
7 P.aeruginosa NBS Neg.
11 P.aeruginosa NBS 4+
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DIAGNOSIS OF BACTERIAL MENINGITIS
ter wasaddedtosupplement the volume of CSF
specimens that were insufficient in volume for
assay. Thedegree ofgelation didnot appear to
be diminished by dilution of these specimens, although the full 70-min incubation was ob-served before
Table 5 shows the total accumulative data from thepresentinvestigationand previous sim-ilar studies (1, 4, 18, 21, 23, 29) evaluating the Limulusassayfor the diagnosisof bacterial men-ingitis. These combined datarepresent a total of393 patients with culture-proven acute bac-terial meningitis, including 321 cases due to gram-negative organisms. If data from all six studies are combined, 92% of all patients with culture-proven, gram-negativebacterial menin-gitiswererapidly detectedby the Limulus test.
Forpurposesofcomparison inthe present study,
only 68% ofcases werecorrectlyrecognized by Gram stain of the CSF. If all cases of
culture-provenbacterial meningitis inthese studies are
considered, theLimulus assaydetected 75% of
cases. The 25% negative rateis duealmost
en-tirely tothe lack of detection of gram-positive
organisms. Onlythe study by McCracken and Sarff (18), involving neonatal meningitis cases, has reporteda
significantnumber of false-nega-tive Limulustestson
patientswith documented meningitis due to gram-negative
Theirfinding of endotoxin in only 71% of CSF samples from infants with gram-negative bacte-rial meningitis is in contrast tothe
The main predictive value of the Limulus
assaylies in its lowpercentageof
reactions (<1%). Any
patientwith a positive CSF Limulus test should therefore be
consid-TABLE 5. Accumulative datafromsixevaluations
ofthe Limulus assayfor diagnosis ofbacterial meningitis
Nofp- No. of pa- No. of pa-No.of pa- tientswith tientswith Study
ptpositiveCSF gram-nega- positiveCSF
tive orga- Limulus
Bermanetal.(1) 107 86 86
Dyson and Cas- 10 6 6
McCracken and 94 84 60a
Sarff (17, 18)
Nachumetal.(21) 43 38 38
Ross etal.(23) 51 38 37
Tuazonetal.(29) 14 8 8
Presentstudy 74 61 61
aNumber ofpatientsderived from author'sstatementthat
"endotoxinwasdetectable inonly71% of CSF samples
ob-tainedontheinitial lumbartap ofinfants with Gramnegative bacterialmeningitis."
eredtohavegram-negative bacterial meningitis
(1).However,anegativeassaydoes not rule out thepossibility of meningitisdue to gram-positive organisms, e.g., infections caused by S. pneu-moniaeorgroupBStreptococcus.
Iftheprevious studies usingCIE (3, 5, 10,11,
20, 27) andthose using the Limulus test (1, 4,
18, 21,23, 29) arereviewed,it becomesapparent
that theoverall abilities of the twoprocedures todetectpatients with meningitis are quite sim-ilar,although for different reasons. CIE suffers from false-negative reactionseither because of toolittle antigen in the CSFtobe detectableor perhaps because ofpoormigration of some an-tigens, such as pneumococcal polysaccharide. The Limulusassay is wholly unable to detect antigens of gram-positiveorganisms, but isvery sensitivetothepresenceof cell wall antigens of gram-negative bacteria.A recent study (9) dem-onstrated thatconcentrationsof bacteriainCSF ofpatients with meningitis ranged from 4.5 x 103to 3x
108organismsperml. Previous inves-tigations (12, 28) have shown thatthethreshold ofdetectability by the Limulusassay basedon the detection ofcell-bound endotoxin is approx-imately 102 to 103organismsperml. Therefore, the
sensitivityof the Limulustestfor the detec-tionofgram-negative organisms in CSF is
There are notyetsufficientdataavailable to
comment on the relative merits of CIE or the
Limulusassayfor thediagnosis of patients with partially orinappropriately treated meningitis. However, thepersistence of antigensin theCSF
for24 to 48hafter the initiation of therapy has
beenreported byusing both methods (3, 10, 18, 21,23). The
prognosticvalue of CIEorLimulus
follow-upCSF specimens during
meningitishasalso not yet been
clarified. It appears that
antigensmay persist in the CSF for 48 h or
CSFcultures. In the current
differed,de-pendingupon the causativeorganisms. In
inade-quatebecause the CSFhadnotbeen sterilized.
72 hor more,
applicationsof the Limulus
as-say have included the demonstration of
plasmaas anindicator of
24)andameasurementof endo-toxin in urine for the detection of bacteriuria (12, 13). The examination of
VOL. 7, 1978 15
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16 JORGENSEN AND LEE
investigators (6, 28). This is dueatleast in part
to the technical problems associated with the
preliminary extraction procedure with
chloro-form or other similar treatments that are
re-quired when plasma proteins are involved.
Fluids, such as CSF or urine, do not require
these extraction procedures and may therefore
be examined by the Limulus assay with much
In the present study, 100% of patients with
gram-negative meningitis were easilyidentified
within 1.5 hby use of theLimulus assay, and
in manycaseswithin30min.Results of Limulus
assays were more easily interpreted andmore
reliable thanwere Gram stain results. The
pri-marydisadvantage of thistechnique is the ina-bility todetect gram-positive organisms, which lackendotoxin.Therefore,theusefulnessof this
testlies intheexamination ofCSFfrompatients
with suspected bacterial meningitis in which
gram-negative organisms are the predominent
agents.These includechildren, patientswhoare
immunodeficient or on immunosuppressive
medications, and patients who have received surgical proceduresortraumatic injuries ofthe
The ability to obtain high-quality Limulus amoebocyte lysatecommerciallynowmakes this
test afeasible adjunctto currentdiagnostic
pro-cedures for bacterial meningitis. However, use
of this testinaroutine hospital diagnostic
lab-oratory requires that theamoebocyte lysate be
licensed by the Food andDrug Administration
hasunfortunatelynotyetbeen obtained. There-fore, Limulus assay of CSF or any body fluid
for thediagnosisof bacterialinfectioncontinues
tobearesearchtechniquethatrequires in most
locales the informedconsentof thepatient.
DifcoL,aboratories, Detroit, Mich.,kindlysupplied L,imu-lusamoebocytelysate (Pyrotest)forthepurposeof thisstudy.
Wethank the staff of the BexarCounty Hospitals' Microbial Pathology L,aboratoryfor theirassistance duringthisstudy.
1. Berman, N.S.,S. E.Siegel,R. Nachum,A.Lipsey,
and J.Leedom. 1976. Cerebrospinal fluid endotoxin
concentrations inGram negative bacterial meningitis.
2.Carpenter, R. R., and R. G. Petersdorf. 1962. The
3. Coonrod,J.D.,and M. W.Rytel.1972. Determination
ofetiologyofbacterialmeningitisbycounter immuno-electrophoresis. L,anceti: 1154-1157.
4. Dyson, D.,andG. Cassady. 1976.Use ofLimulus lysate
fordetectingGram negativeneonatal meningitis.
5. Edwards,E.A., P. M. Muehl,andR.0.Peckenpaugh.
1972. Diagnosisof bacterial meningitis bycounter im-munoelectrophoresis. J. Lab. Clin. Med. 80:449-454. 6. Elin,R.J.,R. A.Robinson,A. S. Levine,and S.M.
Wolf. 1975. Lack of clinical usefulness of theLimulus
testinthe diagnosis of endotoxemia. N. Engl. J. Med. 293:521-524.
7. Feigin,R. D., and P. G. Shackelford. 1973. Value of repeat lumbar puncture in the differentialdiagnosisof meningitis. N.Engl.J. Med. 289:571-574.
8. Feldman, W. E. 1975. Cerebrospinal fluid lactic acid
dehydrogenaseactivitylevels in untreated andpartially
antibiotic treated meningitis. Am. J. Dis. Child. 129:77-80.
9. Feldman, W. E. 19716. Concentirations of baciteia inl cerebrospinalfltuid of patients withbacterialnmeningitis. ,J.Pediatr. 88:549-552.
10. Fossieck, B., Jr.,R.Craig, andP.Y. Paterson.1973. Counterimmunoelectrophoresisforrapiddiagnosisof
meningitisdue toDiplococcus pneumoniae. J.Infect.
11. Higashi,G.I.,J. E.Sippel,N.I.Girgis, and A.
tobacterial culturein thediagnosisofmeningococcal meningitis. Scand.J.Infect.Dis.6:233-235.
12. Jorgensen, J.H.,H. F.Carvajal,B. E.Chipps,and
R. F. Smith. 1973. Rapiddetection ofgram-negative
bacteriuria by use of the Limulus endotoxin assay. Appl. Microbiol.26:38-42.
13. Jorgensen,J.H.,and P.M. Jones. 1974.Comparative evaluationof the Limulusassay andthe direct Gram
stain for detection ofsignificant bacteriuria. Am. J.
14. Jorgensen, J.H., and R. F. Smith.1973.Preparation, sensitivity,andspecificityofLimuluslysate for endo-toxinassay.Appl.Microbiol. 26:43-48.
15. Levin, J.,T. E.Poore,N.S.Young, S. Margolis, N. P. Zauber, A. S. Townes, and W. R. Bell. 1972.
Gramnegative sepsis: detection ofendotoxemia with the Limulus test. Ann.Intern.Med. 76:1-7.
16. McAuley,R.J.,R. D.Ice, and E. G. Curtis.1974.The Limulus test for in vitro pyrogen detection. Am. J.
17. McCracken, G. H., Jr. 1976. Rapid identification of
specificetiologyinmeningitis. ,J. Pediatr. 88:706-708.
18. McCracken, G. H., Jr., and L.D.Sarff.1976.Endotoxin
incerebrospinal fluid: detectioninneonateswith
bac-terialmeningitis. J.Am. Med. Assoc. 235:617-620. 19. Moody,E. E.M.,M. D.Trousdale,J.H.Jorgensen,
andA.S. Shelokov.1975.Bacteriophagesand endo-toxin in licensed live-virus vaccines. J. Infect. Dis. 131:588-591.
20.Myhre,E.B. 1974. Rapid diagnosisof bacterial menin-gitis.Scand. J. Infect.Dis.6:237-239.
21. Nachum,R.,A.Lipsey,and S. E.Siegel. 1973.Rapid detectionofGramnegative bacterial meningitisbythe Limuluslysatetest.N.Engl. J. Med. 289:931-934.
22. Newman, R. B.,R. W.Stevens, andH. A. Gaafar.
1970.Latexagglutinationtest for thediagnosis of
Hae-mophilus influenzae meningitis. J. Lab. Clin. Med.
23. Ross, S.,W.Rodriguez,G.Controni,G.Korengold, S.Watson,and W. Kahn. 1975. Limulus lysate test for Gramnegative bacterial meningitis. J.Am. Med.
24. Rowe,M.I.,D. M.Buckner, andS.Newmark. 1975.
Theearly diagnosisofGramnegative septicemiainthe pediatric surgical patient.Ann.Surg.182:280-286.
25. Schwartz, M. N., and P. R. Dodge. 1962. Bacterial meningitis-a review ofselected aspects. I. General
J. Med. 272:725-731.
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DIAGNOSIS OF BACTERIAL MENINGITIS
26. Severin,W. P. J. 1972. Latexagglutinationin the diag-nosis of meningococcal meningitis. J. Clin. Pathol. 25:1079-1082.
27. Shackelford, P. G., J. Campbell, and R. D. Feigin. 1974. Countercurrent immunoelectrophoresis in the evaluation of childhood infections. J. Pediatr. 85:478-481.
28. Stumacher,R.S., M. S. Kovnat, and W. R. McCabe. 1973.Limitations of theusefulness of the Limulusassay
for endotoxin. N.Engl. J. Med. 288:1261-1264. 29. Tuazon, C. U., A. A. Perez, R. J. Elin, and J. N.
Sheagren. 1977. Detection of endotoxin in cerebrospi-nal and joint fluids by Limulus assay. Arch. Intern. Med. 137:55-56.
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