Rapid diagnosis of gram negative bacterial meningitis by the Limulus endotoxin assay

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0095-1137/78/0007-0012$02.00/0

JOURNALOFCLINICALMICROBIOLOGY, Jan.1978,p.12-17

Copyright ( 1978 AnmericanSocietyforMicrobiology PrintedVol.in7,U.No.S. A.1

Rapid Diagnosis

of

Gram-Negative

Bacterial

Meningitis

by

the Limulus

Endotoxin

Assay

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

with asepticmeningitis,orpatientswithoutmeningitishadnegativeCSFLimulus

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

therapy

isinitiated 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

areurgentlyneeded.

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

techniques

for the detection of these

antigens

(22, 26)

also appear promising. However, both

of these

techniques

require

high-titer-specific

antisera that may bedifficult to obtain

commer-cially.

The determination of the CSF lactate

concentration (8) may alsohelp to

distinguish

bacterial from viralorfungal

meningitis.

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

injectable

pharmaceutical products (16, 19). This test is currently the most sensitive method available

for the detection of endotoxin. The results of

initial studiesconcerningtheuseoftheLimulus

assay for the rapid detection ofgram-negative

bacteria in CSF appear very encouraging (1,21,

23).

Thisstudydescribesourrecentexperience

using

thistechnique for the rapid diagnosis of

gram-negative

bacterial

meningitis.

MATERIALS AND METHODS

Patient group.Adult andpediatricpatients ofthe BexarCountyHospitals,onwhom alumbarpuncture

wasperformedbecauseofsuspectedmeningitis, were

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

0.5ngoflipopolysaccharideperml,whereasPyrotest

coulddetectaslittleas0.06ng/ml.IndividualLimulus

testreactionsonpatient CSFspecimensweregraded

forreactivitywithaschemepreviously described(14).

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

solutionof E.colilipopolysaccharidetoanadditional

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

re-quiredforavalidassayset.

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.

RESULTS

CSF specimens from a total of 305 patients

suspectedofbacterialmeningitiswereexamined

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

withgram-negativebacterialmeningitisyielded

positiveLimulusassays(Table1).

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

patientswhoseCSFsyieldedapparent

false-pos-itiveLimulus assayshadnot receivedprior

an-tibiotictherapy.

Gram stainswerepositiveonCSFsfrom 72%

(44/61)ofpatientswithgram-negativebacterial

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

lum-barpunctureswerenegativein 13instances,and

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

Organismisolated

H. influenzae Neisseriameningitidis E.coli

Klebsiellapneumoniae

Alcaligenesfaecalis

P. aeruginosa

Flavobacterium

men-ingosepticum Acinetobacter

calcoac-eticus var. anitratus

A. calcoaceticus var.

lwoffi

Citrobacterdiversus

GroupBStreptococcus Streptococcus

pneumo-niae

Staphylococcusaureus

Mycobacterium tuber-culosis

Asepticor no

meningi-tis

No.ofpa- Limulus Gram tients positive itaiv

itive

38 38

6 6

2 2

1 1

4 4

1 1

30 5 3

0

1 2 1

1 1 0

1 1 1

4 0 2

6 1a 3

3 0 1

1 0 0

230 2 0

aGraded

as

1+

reaction;

see

text.

13

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14 JORGENSEN AND LEE

TABLE2. Patients withpersistentCSF endotoxin

during antibiotic therapy

Limu- Repeat Repeat Organism isolated _patientsNo. of lus_itivepos- _positiveculture _staingram

pos-itive

H.influenzae 13 13 1 1

E.coli 3 3 3 1

Neisseria meningi- 1 1 1 0

tidis

Klebsiella pneumo- 1 1 1 0

niae

Flavobacterium 1 1 1 0

meningosepticum

P.aeruginosa 2 2 2 0

Acinetobacter cal- 1 1 1 0

coaceticusvar.

anitratus

tive Gram stainfrom the follow-up CSF

speci-men.

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

beseenfromthesedata,itwasnotunusual for

patients with sterile repeat CSF specimens to

haveapersistence of endotoxin 24to72hafter

the initiation of antibiotic therapy, with one

patient demonstrating persistent CSF endotoxin

for 9days.

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

defi-cits.

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

wasobtained.

DISCUSSION

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

inbacterialmeningitis. However,organismsnot

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

amoebocytelysates preparedinourlaboratory.

Whenmorethan 100assayswereperformedin

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

as-sayresult

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

assayswerenegative

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.

11 P.aeruginosa NBS 4+

aGNR, Gram-negativerods.

bNBS,Nobacteriaseen.

cNeg.,Negative.

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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

examining

thelysatetubes.

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. Only

the study by McCracken and Sarff (18), involving neonatal meningitis cases, has reporteda

significant

number of false-nega-tive Limulustestson

patients

with documented meningitis due to gram-negative

organisms.

Theirfinding of endotoxin in only 71% of CSF samples from infants with gram-negative bacte-rial meningitis is in contrast tothe

findings

of

thecurrent

study

andthosepreviously (1,4,21,

23,29).

The main predictive value of the Limulus

assaylies in its lowpercentageof

false-positive

reactions (<1%). Any

patient

with 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 patientswith tientswith Study

ptpositive

CSF gram-nega- positiveCSF

tive orga- Limulus

as-u

msms

inCSF says

Bermanetal.(1) 107 86 86

Dyson and Cas- 10 6 6

sady (4)

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

108

organismsperml. 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

sensitivity

of the Limulustestfor the detec-tionofgram-negative organisms in CSF is

easily

explained.

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

prognostic

value of CIEorLimulus

assay on

follow-up

CSF specimens during

ther-apy for

meningitis

hasalso not yet been

fully

clarified. It appears that

antigens

may persist in the CSF for 48 h or

longer, despite

sterile

CSF

cultures. In the current

study,

the

impli-cation of

persistent

CSF endotoxin

differed,

de-pendingupon the causativeorganisms. In

non-H.

influenzae meningitis,

the

persistence

of endotoxin

suggested

that

therapy

was

inade-quatebecause the CSFhadnotbeen sterilized.

However,

with H.

influenzae meningitis,

endo-toxinpersistedinthe

CSF

ofsome

patients

for

72 hor more,

although

viable

organisms

could

nolongerberecovered.

Prior clinical

applications

of the Limulus

as-say have included the demonstration of

endo-toxin in

plasma

as anindicator of

gram-negative

septicemia

(15,

24)

andameasurementof endo-toxin in urine for the detection of bacteriuria (12, 13). The examination of

plasma

for

endo-toxin hasnotbeen

uniformly

successfulwithall

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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

greaterfacility.

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

centralnervoussystem.

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

asaninvitrodiagnosticproduct. Thislicensure

hasunfortunatelynotyetbeen obtained. There-fore, Limulus assay of CSF or any body fluid

for thediagnosisof bacterialinfectioncontinues

tobearesearchtechniquethatrequires in most

locales the informedconsentof thepatient.

ACKNOWLEDGMENTS

DifcoL,aboratories, Detroit, Mich.,kindlysupplied L,imu-lusamoebocytelysate (Pyrotest)forthepurposeof thisstudy.

Wethank the staff of the BexarCounty Hospitals' Microbial Pathology L,aboratoryfor theirassistance duringthisstudy.

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VOL. 7, 1978