Use of Limulus Lysate for Detecting Gram-Negative Neonatal Meningitis

(1)

Use of Limulus

Lysate

for Detecting

Gram-Negative

Neonatal

Meningitis

Donald Dyson, M.D., and George Cassady, M.D.

10)111 tluc I)ici.sion of Pc’rinata! ‘ile(lictfle, Department of Pediatrics, University of Ala!)a;na, Birmingham

ABSTRACT. The Linuuh,.s lvsate test was evaluated as a

niethod for rapid detection of neonatal gram-negative

bacte-rial meningitis. A total of 208 CSF samples were collected

froni 145 newborn infants suspected of having meningitis.

Initial samples fronl all six 1)abies with culture-proven

gram-negative bacterial meningitis had positive Limulus tests

within 30 niinutes of incul)ation. Samples from 14 infants

with gram-negative organisnls isolated only in blood and/or

urine .as well as from four neonates with gram-positive

organisnis in CSF cultures and from 13 with gram-positive

organisms in blood cultures all yielded negative Liniulus

tests.

Thus, of 37 newborn infants with bacteria demonstrated in

systemic cultures, only those six with gram-negative

organisms ill CSF had positive CSF Liunuhis tests. The CSF

Li,iuuulus test was shown to be a rapid, reliable, and specific

test for the detection of neonatal gram-negative bacterial

nieningitis. Pediatrics 58: 105- 109, 1976, MENINGITIS,

(;RAM-NE(;ATIVE BACTEBIA, Linuuilus LYSATE TEST.

Bacterial

meningitis

in newborn

infants

differs

from

that in older infants and children by its paucity of clinical manifestations,I2 greater

frequency

of gram -negative organisms, ‘

and

high Illorbidity and mortality.’’T” In addition, CSF gram stain is, on occasion, not useftil due to

the

small

numbers

of bacteria

and

WBC

present

early in the disease process.’’’ Early, precise diagnosis of meningitis, therefore, remains a

currently unsolved problem in the newborn infant.

Levin

and

Bang’

have

shown

that

a

lysate

prepared

from

the

amebocytes

of

the

horseshoe

crab,

Limulus

polyphenius,

will

undergo

gelation

when

exposed

to minute

amounts

of

gram-nega-tive

endotoxin.

This

reaction

has

been

developed

as the basis for a simple, rapid method for

detecting

the

presence

of

endotoxin

in

various

body

fluids.’42’

In the

present

report,

this

test

has

been

evaluated

as a method

for

rapid

detection

of

gram-negative bacterial meningitis in 145

new-born

infants.

MATERIALS

AND

METHODS

A total of 208 CSF samples were collected from 145 neonates with suspected meningitis during a seven-month period. In addition, 190 blood

cultures

and

156 suprapubic

urine

cultures

were

concurrently

obtained.

Small

aliquots

were

obtained

for

Limulus

testing

and

routine

chemi-cal,

bacteriologic,

and

gram-staining

studies

were

carried

out

on

the

remainder

of the

CSF

sample.

The

Limulus

test

aliquot

was

collected

in sterile,

pyrogen-free,

screw-cap

culture

tubes

which

had

been

heated

in a dry

oven

at

300 C

for

a minimum

of three

hours;

the

samples

were

stored

at -20

C

until

tested.

A lyophilized

Liniulus

amebocyte lysate,

prepared

by the

method

of Levin

and

Bang,’4

was

reconstituted

with

sterile

water

for

injection

(Received September 2; revision accepted for publication

November 4, 1975.)

Supported by the Perinatal Infections and Immunity Grant

and training grant HD 00413-03 from the National Institute

of Child Health and Human Development.

ADDRESS FOR REPRINTS: (G.C.) Division of Perinatal

Medicine, Department of Pediatrics, University Station,

(2)

TABLE I

RELATIONSHIP OF CSF Limulus TEST TO BACTERIAL CSF CULTURES AVFER 30 MINUTES IN 145 NEWBORN INFANTS

Limulus Test Results

Organfsin isolated From CSF

No.of

infants

r-Negatice + 1 + 2

-‘

+ 3

E.coli 5 0 0 2 3

Serratia niarescens 1 0 0 0 1

Staphylococcus aureus 2 2 0 0 0

Group B Streptococcus 1 1 0 0 0

Staphylococcus epidermklis 1 1 0 0 0

None 135 132 3 0 0

(Invex) and stored at -20 C until used. All lots of

lysate

were

sensitive

to

0. 1 ng/ml of

Salmo-nella

typhosa

lipopolysaccharide

B (Difco,

0901).

All glassware used, including pipettes and culture tubes, was heated in a dry oven at 300 C for a minimum of three hours.

The

Limulus

test

was

performed

according

to

the technique of Levin et al22’

with

two

modifications: (1) chloroform extraction

unneces-sary

for testing of CSF’3 was not employed, and

(2)

reduced volumes of CSF and lysate (0.05 ml

each)

were

used.

The

tubes

were

inspected

at

30-minute intervals during a total two-hour incuba-tion and results were recorded using the following

scale:

negative,

no

change

in viscosity

or

turbid-ity;

+ 1, slight flocculation with no change in

viscosity;

+ 2, increase in both viscosity and

turbidity; and + 3, solid gel. Positive and negative

controls

were

included

with

each

test.

S.

typhosa

lipopolysaccharide B (0.05 ml of solution with a

concentration of 0.1tg/ml of pyrogen-free water)

used

as a positive

control

uniformly

gave

a

+3

reaction within 30 minutes. Sterile water for

injection

(0.05

ml

of

Invex)

was

used

for

a

negative control. All tests were interpreted by one author (D.D). Culture results and patient

identification

were

unknown

at time

of

interpre-tation.

RESULTS

The relation of the

Limulus

test

to initial

CSF

culture results is shown in Table I. Six newborn infants with culture-proven gram-negative

bacte-rial

meningitis had a + 2 or + 3 test within 30 minutes and all had a + 3 test within one hour of incubation. In contrast, four babies with

gram-positive

organisms

isolated

from

their

CSF

had

negative

Limulus

tests

after

two

hours

of

incuba-tion. All infants with sterile CSF had a negative

(132

patients)

or

+ 1 limulus test (3 patients) at 30

minutes; none of these + 1 tests progressed with incubation for two hours.

All patients with organisms isolated from CSF

also

had

positive

blood

cultures

for

the

same

genus of organism. In 27 patients, however,

organisms

were

grown

from

blood

(24)

or urine

(3)

but

CSF

cultures

were

negative.

In

all

of

these

babies,

Limulus

test

was

negative

after

two

hours

of incubation (Table II).

Three of the six patients with gram-negative

meningitis

died

within

one

hour

of

the

initial

lumbar

puncture.

One

of

the

three

surviving

babies

was

treated

with

an

intrathecal

antibiotic

and had CSF sterilization and a negative

Limulus

test

within

24 hours

of treatment.

The

other

two,

treated only with parenteral antibiotics, had

persistently

positive

(+ 3) Limulus

tests

two

and

four

days

after

bacteriologic

sterilization

of

the

CSF. All three surviving neonates had negative CSF cultures and negative Limulus tests after

three

weeks

of antibiotic

treatment.

DISCUSSION

Despite

the

development

and

use

of

new

antimicrobial agents in recent years, mortality

and

morbidity

of neonatal

meningitis

continue

to

be high’’T’ with

Escherichia

coli24

and group B

/3-hemolytic

Streptococcus4

the

most

common

single

organisms.

A

rapid,

reliable

method

of

determining

the

etiologic

agent

would

allow

earlier

institution

of appropriate

antibiotic

ther-apy.

The traditional method for rapid

identifica-tion

of infecting

agent,

the

gram

stain,

is

maccu-rate in 20% to 40% of cases.’ “ Despite

modifica-tions,25

technical

difficulties

to

interfer-ence

by

drugs,2’ certain disease and an

increased

neutrophil

reduction

by neonatal

leuko-cytes in the newborn2 :10 :13

interfere

with

accurate utilization of the nitroblue tetrazolium

(3)

TABLE II

RELATIONSHIP OF CSF Limulus TEST TO BACTERIAL ISOLATIONS FROM SYSTEMIC CULTURES IN 27 NEWBORN INFANTS

Organisms

No.

Patie

of

nts Site of isolation

No. of

Patients CSF Limulus Test

No. of

Patients

E. coli 9 Blood 6 Negative 9

Urine 3

Kiebsiella pneunzoniac 1 Blood 1 Negative 1

P. aeruginosa 1 Blood 1 Negative 1

S. inarescens 2 Blood 2 Negative 2

Proteus mirabilis 1 Blood 1 Negative 1

S.aureus 2 Blood 2 Negative 2

Enterococcus 1 Blood 1 Negative 1

S.epidermidis 3 Blood 3 Negative 3

Streptococcus ciridans 2 Blood 2 Negative 2

Group B Streptococcus 1 Blood 1 Negative 1

Diphtheroides 3 Blood 3 Negative 3

Candida albicans 1 Blood 1 Negative 1

clinically

pertinent

information

regarding

the

etiologic

agent.

In 1946, Bang demonstrated that a liquid material, prepared from the amebocytes of the

horseshoe

crab,

would

gel

when

exposed

to

bacte-rial

endotoxin.”

Since

that

time,

Levin,

Bang,

and

others

have

described

the

preparation

of a lysate

from

the

amebocytes

of the

horseshoe

crab

which

undergoes gelation in the presence of minute

amounts

of endotoxin

produced

by gram-negative

bacteria.’’

Many

studies

have

subsequently

shown that the Limulus

test

is a rapid,

sensitive,

and

reproducible

method

of

detecting

bacterial

endotoxin

in

drugs,

fluids,

and

other

agents

prepared

for

parenteral

administration.2’’’

The

clinical

application

of this

test

for

detection

of

endotoxin

in

blood,

however,

has

met

with

mixed

results.

Several

series

have

noted

a high

correlation

between

a positive

Limulus

test

and

positive

blood

cultures

for

gram-negative

orga-Other authors, however, have

reported

a high

incidence

of

false-positives

and

false-negatives

as well

as problems

of

nonspecific-ity.’4

‘

Causes

for

these

discrepancies

which

have

been

identified

include

endotoxemia

of intestinal

origin,’M2

localized

infections

producing

endo-toxemia without bacteremia,22 variations in

extraction

of inhibitor

factor(s)

in

blood,14I7.3i

and

platelet-binding

of endotoxin.54

Data

from

dogs55

suggest

that

the

blood-brain

barrier

is

impermea-ble to endotoxin. so, circumvention of the many

technical

problems

using

blood

for

the

Limulus

test is theoretically possible by examining CSF in

the

patient

with

meningitis.

Data from the present study support this

possi-bility.

In

145 newborn

infants

with

suspected

systemic

infection,

those

six

with

gram-negative

meningitis

all had

a positive

CSF

Limulus

test.

In

contrast,

initial

routine

CSF

gram

stains

revealed

gram-negative

bacteria

in only

three

of these

six

patients.

All

31 babies

with

gram-positive

organisms

in CSF

or with

organisms

isolated

only

from blood or urine had negative tests. In the

present

series,

therefore,

neither

false-negatives

nor

false-positives

were

found.

These

observa-tions,

coupled

with

similar

data

from

38 infants

and children recently reported,” suggest that the CSF

Limulus

test

is a reliable

and

specific

test

for

gram-negative bacterial meningitis. Although the variety of gram-negative organisms in the present

study was limited, previous reports suggest that

meningitis

caused

by

genera

of the

family

Enter-bacteriaccae, in addition to

Ltemophilus

influen-za,

Neisseria

meningitidis,

Pseudomonas

aerugi-nosa,

or

Bacteroides fragilis’35221’4#{176}414 would

also

be

detected

with

this

test.

The

ability

to

diagnose

neonatal

meningitis

with

these

or-ganisms

with certainty within 30 to 60 minutes of

lumbar puncture affords obvious therapeutic

advantages.

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Cu DQ, et a!: Bubonic plague:

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(5)

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Lal) (Jill Med 75:903, 1970.

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ACKNOWLEDGEMENTS

We express appreciation to the Pediatric Housestaff and

Nursing Staff of the University of Alabama in Birmingham

Newborn Intensive Care Unit, without whose cooperation

the vork WOLIld have been im1)OSSible. \Ve also thank Dr.

Ilavniond Bobo for his invaluable l)acteriologic assistance,

aiid Miss PenI’ly Shook for her secretarial help.

H.E.W.

ALTERS

SCOPE

OF SPANKING

ORDERS

\iVashington, Jan. 17 (UPI)-Apologizing for “any inconvenience caused,”

the

Department

of FIealtl#{236},

Education and \Velfare said Saturday it was sharply

reducing the scope of a student s)anking ordei issued last summer to the

nation’s public schools.

The

original

order

Ieqllircd all

16,000

United States public school systems to

keep

detailed

records

on

student

punishment.

In softening that directive. the department said extensive record-keeping

now

will

be re(1uired

on1’ at the 3,00() elementary and high-school systems that

have

90 percent

of

the nation’s niinority students and will not take effect until

fall.

At the same time, however,

Il.E.\\

‘T increased

the

complexity of other

disciplinaiy records iequired at all schools receiving Federal aid-a category

covering

most

of the

16,000

p111)lic

districts

and

some

private

schools.

Those

less-detailed

i-equii-ements called for numerical statistics on the race

and

sex

of students

disciplined

to help

in enforcing

laws

prohibiting

Federal

aid

to schools

that

practice

cliscriniination.

\Tetv h)rk Times

(6)

1976;58;105

Pediatrics

Donald Dyson and George Cassady

Lysate for Detecting Gram-Negative Neonatal Meningitis

Limulus

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