Rapid diagnosis of lymphocytic meningitis by frequency pulsed electron capture gas liquid chromatography: differentiation of tuberculous, cryptococcal, and viral meningitis

Copyright C)1977 American Society forMicrobiology Printed in U.S.A.

Rapid Diagnosis of Lymphocytic

Meningitis by

Frequency-Pulsed Electron

Capture Gas-Liquid Chromatography:

Differentiation

of Tuberculous;

Cryptococcal, and Viral

Meningitis

ROBERT B. CRAVEN, JOHN B. BROOKS,* DAVID C. EDMAN,1 JAMES D. CONVERSE,' JOHN GREENLEE,2 DAVID SCHLOSSBERG,3 THOMAS FURLOW,2 JACK M. GWALTNEY, JR.,I AND

WALTER F. MINER'

Centerfor DiseaseControl, Public HealthService, U.S. Department of Health, Education,and Welfare, Atlanta,Georgia30333

Received for publication7March1977

Cerebrospinal fluid specimens from patients with tuberculous (17 cases),

cryptococcal (15 cases), and viral (14 cases) meningitis were analyzed by

fre-quency-pulsed electroncapturegas-liquid chromatography andmass

spectrome-try.Compounds that disappeared after therapywerefoundtobepresentineach

ofthese specimens andwerenotdetectedincontrols. They occurredin repetitive

patterns such that these three types of meningitis could be rapidly

distin-guished. The compoundassociated with tuberculousmeningitishasbeen

tenta-tively identified. These findings have implications for rapid diagnosis,

patho-physiological studies, and possiblenewtherapeutic approaches.

Gas-liquid chromatography (GLC) has been

usedtodifferentiatevariousspeciesof bacteria

by analysis of theirmetabolicproductsinspent

culture mediaorof theircell wall constituents

(6, 8-11, 12, 19-24). More recently, the

tech-nique has been applied to the study of body

fluids during infection (2, 6a, 7, 25; J. B.

Brooks, R. B. Craven, D. Schlossberg, C. C.

Alley, and F. M. Pitts, submitted for

publica-tion). Schlossbergetal., using GLC

techniques

developed

inthislaboratory, demonstrated the

possible rapid diagnosisofcryptococcal

menin-gitis (25). The difficulty in obtaining timely laboratory confirmationof theagent

producing

lymphocytic meningitis, e.g., cryptococcal,

tu-berculous,

or

viral, prompted

the present

in-vestigationof GLC as arapid etiological

diag-nostic method.

MATERIALS AND METHODS

CSF specimens. All specimens were the excess

left after routine laboratorystudieswereperformed. Control specimens consisted of the excess cerebro-spinal fluid (CSF) from myelograms or spinal epi-duralanesthesia andwerebacteriologicallyculture

negative, with normalcellcount, glucose, and

pro-tein.

Twelveculture-positive specimens frompatients

IPresentaddress: U.S. Naval Medical Research Unitno.

3,FPO, NY09527.

:2Presentaddress:University ofVirginiaSchool of Medi-cine,Charlottesville,VA 22904.

3Presentaddress:Emory UniversitySchoolofMedicine, Atlanta,GA 30322.

withtuberculous meningitis were obtained from the meningitis research ward of the U.S. Naval Medical Research Unit no. 3 (NAMRU-3), Cairo, Egypt. Seven samples from cryptococcal meningitis

pa-tientswerecollected by us, and an additional eight sampleswereprovided byLeoKaufman'slaboratory

atthe Center forDiseaseControl (CDC)inAtlanta, Ga.Theseeight samples were from specimens sub-mitted by the University Collaborative Study of Cryptococcal Meningitis to be analyzed for crypto-coccalantigen. Allcryptococcal specimens were cul-turepositive orantigenpositive orboth.

We collected five specimens from patients with clinically diagnosed aseptic meningitis, three from

oneclustered outbreak and two from another. We

also obtainedCSFfrompatients withbiopsy-proven herpes simplexencephalitis andtwospecimens from patients with encephalitis associated with herpes

zoster skin infection of the face. Anumber of cul-ture-documented specimens of viralmeningitis and encephalitis wereprovided by Richard Emmons of the CaliforniaDepartment of Health.One specimen was from a patient with toxoplasmosis meningitis (14).

Apparatus. APerkin-Elmer gas chromatograph, model 900, equipped with a 63Ni 10-mCi frequency

pulse-modulated electron capture (EC) detector,

switching valves, and a25.4-cm potentiometric re-corder,wasused.The instrumentwasoperatedwith

twocoiledglass columns (0.3 cm in IDby7.3m in

length). One column (nonpolar) was packed with Chromosorb W, 80/100 mesh (AW-DMCS H.P.),

coated with 3% OV-1 (Applied Science

Laborato-ries).Thesecondcolumn(polar)waspackedwith3%

Dexsil410 (Analabs). Theswitchingvalves

permit-ted a comparative analysis of the effluent from 27

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28 CRAVEN ET AL.

either column through asingle detector. Reagents

and instrument settings were as previously

pub-lished(1, 3,4). A Perkin-Elmer PEP-1 datasystem with Modular Software 2, Revision C, was usedto

refine dataandtotentatively identify compounds by

their retention times, as previously determined

fromanalysisofstandard mixtures(5).

Derivatization procedures. Derivitization

proce-dures have been described in detail elsewhere(1, 3,

4). Toexploitthespecificityof the ECdetector, we

formedelectron-capturingderivatives ofacids,

alco-hols, and amines before GLC analysis as follows

(15). A 2-ml specimen of CSF was acidified to

ap-proximately pH 2 by adding0.1 ml of 50% H2SO4.

Theacidified samplewasextractedby shakingwith 20mlof6%ethanolinchloroformtoobtain acids and alcohols. Thechloroform-ethanolwasremoved,and

the samplewasmade basic (pH 10)and re-extracted

for amines with 20 ml of chloroform. ThepH2and

pH 10fractionswereput into separate 50-ml

beak-ersandevaporatedtoabout 1 ml with astreamof

clean dryair.Thesamplesweretransferredto12-by

75-mmtesttubes withdisposable Pasteurpipettes;

anyvisiblewaterlayerwasdiscarded.

Sampleswerefurtherdriedby adding 100mgof

MgSO4tothe concentrate andshaking. Next, they

were centrifuged, and the solvent layer was

re-moved to a clean 12-by 75-mmtesttube. The

re-mainingMgSO4waswashed with1mlof chloroform

andcentrifuged; thechloroformlayerwasdecanted

andcombined with thepreviouslydecanted solvent. TheMgSO4 wasdiscarded. Thesamples were

con-centrated undera gentle stream of airto

approxi-mately0.1ml.

Acids and alcoholspresentinthe pH2 fractions

weretreatedwithtrichloroethanoland

heptafluoro-butyric and anhydride (HFBA) to form

electron-capturing derivatives as follows: 1 drop offreshly

prepared trichloroethanol-chloroform (5:95)solution

and 3 drops of HFBA were added to each pH 2

fraction, and these fractions wereallowedto stand atroom temperature for 30 min. Then, 0.1 ml of

chloroformand4drops of 0.1 N HCl wereaddedto each sample, whichwas shaken and allowedtosit for 3min. Theaqueouslayerwasremoved and

dis-carded,and4dropsof 0.1NNaOHwasaddedtothe organic layer. Thiswasshaken and allowedtositfor 3min. Theorganic layerwasremovedtoaclean

12-by 75-mmtesttube andconcentratedto near

dry-ness. Next, 0.1 ml of xylene was added, and the

samplewasagain evaporatedtoneardrynessinan

80to90°Csand bath underastreamofclean dry air. A second 0.1 ml of xylene was added as the final

solvent.

Two microliters of each samplewasthen injected

intotheGLC column. The columnoventemperature

wasincreased at4°C/minfrom an initial tempera-tureof110°Ctoafinaltemperatureof245°C,where itwasheldfor30min. Under theseconditions, acids varying in length from2to20carbonatomscould be detected fromasingle derivatization procedureand chromatographic run (4). Recently, we have found

that increasing the amount of HFBA from 3 to 6 drops resultsinanincrease in theamountof deriva-tiveformed.

Amines presentinthepH10extractionwere deri-vatized as follows. To the sample remaining after

the MgSO4 drying step was added 1 drop ofa 1:3

mixture of pyridine-chloroform and 6 drops of HFBA.Thismixture wasevaporated by airtoabout

0.5 drop; the tube wasthen stopperedwith a cork and heated for4min inboilingwater.The tubewas

cooled and 0.1 ml of chloroform was added. This mixture waswashed with4drops each of0.1NHCl and0.1NNaOHasdescribedabove, except that the sample was allowedto sitin the basic wash for30

min. Thechloroform layer from the basic washwas

removedtoa cleandrytesttubeandevaporatedto

neardrynessundera streamof cleandry air, and0.1

ml of ether was added as a final solvent for GLC analysis. Two microliters of each sample was

in-jected,beginningat90°Cfor8min,and thenraised

to 225°C at 4°C/min, where the temperature was

held isothermally for 30 min. Total analysis of aminesoracids andalcohols requiresapproximately 3h.

RESULTS

The 12 samples from patients with proven

tuberculousmeningitisintheearly stagewere

remarkablysimilar in boththeamines detected

inthe HFBA derivative fromthepH10extract

and in the acids and alcohols detected in the

trichloroethanol derivative from thepH2.0

ex-tract. The samepatternwas found in CSF from

fiveclinically suspected cases. Figure 1 shows

chromatograms obtainedfrom the CSF of four

different patientswithculture-proven

tubercu-lous meningitisby usingthe pH 10 HFBA

de-rivative. Figure 2 shows a fifth patient with

tuberculous meningitis whose CSF

demon-strates the differences noted between

speci-mensobtainedduring theacutephaseof illness

(Fig. 2A) and after successful treatment (Fig.

2B). The presenceof severalcompoundsinCSF

obtained early in the course of tuberculous

meningitis contrastssharply with their virtual

absence both in successfully treated patients

(Fig. 2B) andinthe uninfected controls.

Peaks with the same numbers in Fig. 1A

through D have the sameretention times and

maybeusedtostudy similaritiesinthe

profiles

ofpatients. Peaks numbered 1 through7 were

commontoall tracings;however, peaks6and7,

which were present in small amounts in the

original chromatogram, were notvisible in the

reduced tracing (Fig. 1B) and hence are not

labeled in that tracing. The most important

peak in these tracings is labeled KHI. It has

been identified by mass spectrometry as

3-(2-ketohexyl)indoline. Data supporting this

iden-tificationwill bereported elsewhere (6a).

KHI has been found in every case of acute

tuberculous meningitis but not in the other

types ofmeningitis studied. In serial CSF

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29

RETENTION TIME (MINUTES)

225°

TEMPERATURE

FIG. 1. HFBAderivativesof the pH10extractions of CSF from four patients with meningitis due to

Mycobacterium tuberculosis. Thepeaklabeled KHI has been identified by mass spectrometry as 3-2-ketohexyl)indoline and is characteristic of tubercu-lous meningitis.

mens of fivepatients, KHI disappeared slowly over the first 2 months oftherapy; however,

when steroid therapy was used, it tended to

disappear more rapidly. Fig. 1D represents a

partially treatedcase,and the reducedamount

ofKHI present isevident. Figure 1C was

ob-tained froma1.5-mlsample ofCSF.Theeffect of reduced samplesize onthe profileingeneral,

as well as onKHI, maybe seen. The value of

this compound in distinguishing tuberculous

from cryptococcal and viral infections maybe

seenby comparing Fig. 3A tothe other tracings

inthefigure. BynotingthepresenceofKHI as partof the

typical

patternshownin Fig. 1,we

have been abletoidentify correctlytwo casesof tuberculous meningitis before the culture

re-sults were available from NAMRU-3.

Two patterns ofcryptococcal meningitis are

encountered in the pH 10 extraction (Fig. 3B

and C). The occurrence of the B and I or J

peaks, however, distinguished this disorder

from viralmeningitisinthe15samples studied.

The type IIpattern (Fig. 3B) is the same as that

previously found by Schlossberg et al.

(25)

in thislaboratory and was present in 7 of the 15

specimens studied. ThetypeIpattern contains twodifferent compounds, labeledA and I. We

suspectthat the occurrence ofthe type II

pat-tern maybeduetolesserseverity ofdiseaseor

to the specimen being obtained early in the

treatmentcourse. However, inspecimensfrom

two acute culture-positive untreated cases, a

typeII pattern wasobtained. Figure 2C andD

show the effects oftreatment with

amphoteri-cinB onthe CSF ofapatient withcryptococcal

meningitis. Figure2Cwasfrom aCSFobtained

earlyinthe treatment course,

and

it is atypeII

pattern. Figure 2Dshows anormalpattern in

thispatientafter6weeks of therapy.

Afurtherpossible cause for the two patterns

may be real metabolic differences among the

three serogroups of Cryptococcus neoformans.

To study this, we inoculated serogroups A, B,

and C intoneisseriadefined medium and

ana-lyzed the spent culture medium for metabolic products, using the procedures described

ear-lier(19). Serogroup Adidnotproduceanamine

thatwasproduced byBand C. Serotype C did

not produce five acids that were produced by both A and B. Thus,

metabolic

differences do existbetween serogroups.

Figure 3DandE demonstrate the major

pat-Patient #2497

Acute tuberculous

meningitis

Patient #2497

w \ After 6months of therapy

cn \fortuberculosis meningitis

w JG-H

w \ Cryptococcalmeningitis

earlytreatment phase

90°C 225°C

FIG. 2. Effect oftreatment on acute tuberculous

and cryptococcal meningitis. HFBA derivatives of

amines inCSFofapatient withacute(A) and treated (B) tuberculous meningitis and ofa patient with

acute(C) and treated(D)cryptocococcal meningitis.

BandD arelike normal CSF. VOL. 6, 1977

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Thispatternrecurred with the exacerbationsof

KHI thispatient's illness, which hasbeen described

Acutetuberculous in detail elsewhere (14). The MNO

complex

of

meningitis peaks distinguishes this type of meningitis

A from the othertypes studied.

A further demonstration of differences in

l1J viral cases may be seen in Fig. 4, which

com-DS-14 pares the pH 2 derivatized extract ofsamples

N

AD

\ Cryptococcal meningitis obtained from patients in two separate

clus-K ~~~~~TypeIIPattern

B tered outbreaks of clinically diagnosed aseptic

meningitis, one patient with biopsy-proven

herpes simplex encephalitis, and two patients

Xd)

l

XIJG-H

with herpes zoster-associated encephalitis. The

z rAIH lj t PCryptococcolmeningitis patterns are easily

distinguishable

by visual

R~~ ~ ~ ~~~Tp atrnarditnusbl

inspection

ofthe

chromatograms.

Qu I K | Herpessimplex

DISCUSSION

l,,encephalitis Thetheoretical and

practical

considerations

D involved in applying GLC to the analysis of

infected body fluids were discussed by Cherry

and Moss in 1969 (13).

Recently,

Brooks has

Echo 4meningitis reviewed advances in methodology and

equip-E_

Jt ment which have established the feasibility of

_

E this

approach

to

diagnosis

(J.B. Brooks,

Micro-JG-B

biology-1975,

p.

45-54,

American

Society

for

Acute

Toxoplosmcgondii

Microbiology, Washington, D.C., 1975). Previ-meningitis

ously,

Mitruka etal.

suggested

thatthe

inter-J No action of the host and infecting agent could

F produce compounds whose detection by GLC

5 10 15 20 25 30 35 40 45 5'5 60 65 70 75 o085 mightleadto arapid

diagnostic

technique

(17).

___-TIME---

TIME The implications and desirability of this

ap-900C 225'C proachhavebeendiscussedrecently by

Schloss-FIG. 3. Comparison ofamine patterns associated

with tuberculous, cryptococcal, viral, and toxo- AsepticMeningitis

plasma meningitis. The distinctive compound

associ-ated with tuberculous meningitis,

3-(2'-ketohexyl)-indoline, may be seen in tracing A (peak labeled

KHIL.A

ternsassociated with viral central nervoussys- AsepticMeningitis

tem (CNS) infections.

Chromatographic

peaks

a-ofimportance for distinguishing viral from C. rB

neoformans infection are under brackets in _

Fig. 3B through E. Further, the viral patterns

,l

HerpesiSimple

(Fig. 3D and E) do nothave peaks B, I, orJ, Encephalitis

which were present in cryptococcal meningitis,

(lcoses)

but

they

do have peaks 1 or 1and 2 plus the C

FGH complex.

Although the distinctive FGH complex is Herpes Zoster

consistent in the 14 viral specimens studied to Encephlitis

date, different patterns do seem to be associated

with different viruses. This may be seen in D

5 1051~2025303'54'0 5 50~560656 70i58084590

Figure 3D, obtained from apatient with herpes T

simplex encephalitis.

Peak Landthe unlabeled 145;dC --245°C

peak precedingKare notpresent in the sample

FIG.

4. GLCprofiles ofvarious viral CNS

infec-from an ECHO-4 meningitis patient (Fig. 3E).

tions (pH

2

extraction).

Trichloroethanolderivatives

Figure 3F shows the amine pattern associated of acids and alcohols detected in viral meningitis

with a case ofToxoplasmagondii meningitis. andencephalitis.

30 CRAVEN ET AL. J. CLIN. MICROBIOL.

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RAPID DIAGNOSIS OF MENINGITIS BY EC GLC

bergetal. (25).

That distinctive compounds are present in

the course of infection and not in the same

patient after treatment or in normal controls

hasbeendemonstratedin thisstudy, as well as

in studies of cryptococcal meningitis (25),

tu-berculousmeningitis in primates (J. B. Brooks,

L.

Thacker,

R.

Broderson,

and E. R. Beam,

unpublished data), andseptic arthritis (7).

Us-ing adifferent GLC technique,Amundsonetal.

havealsoshownchanges occurringin response

tocryptococcal and other meningitidies (2). The

presence ofalarge numberofcompounds

dur-inginfection andtheabilityto detect them by ultrasensitive GLC techniques offer

possibili-ties for future investigations.

The origin of these compounds detected by

GLC, whether from infected hostcells,

inflam-matory response, or metabolic products of the

organismitself, is unknown.

Schlossberg

etal.

showed that C. neoformans grown in vitro in

normal human CSF produced a chromato-graphic pattern similartothatdetectedin

nat-urally infected CSF (25). In the case of viral

infection, however, it isreasonable to assume

that thecompoundsarefromtheinfectedtarget

tissue or associated

inflammatory

cells.

What-everthe origin of these compounds, the possi-bility of theirbeing involvedinthe pathophysi-ologyofmeningitisis animportantquestion for further study. If

they

are shownto be

impor-tant inthe diseaseprocess, then anew

neuro-pharmacological approachto therapy

might

be

possible.

The compound KHI found in tuberculous

meningitis is of interest because ofits

struc-tural similaritiesto

indolic-type compounds

of neurochemical importance, suchasmelatonin.

The amine derivatization procedure described above does not derivatize

hydroxy-substituted

indolic compounds, such as serotonin and

5-hydroxyindoleaceticacid. Thismaybearesult

ofdegradation of these

compounds

atthe

high

pH used during extraction (pH 10).

Thus,

we

may assume that amines of this type are not

producing the peaks noted in the

chromato-gramsofFig. 3. Ultimate determination of the

structures of these

compounds

by

mass

spec-trometry willrequire

obtaining

themingreater

concentration, since the EC detector is much

more sensitive than the massspectrometer for

these types ofderivatives. This

difficulty

may

be overcomeby trapping the

compounds

as

they

pass

throughthe EC detectoror

by extracting

much larger samples for CSF for

subsequent

analysis bymass spectrometry.

In agreement with the

findings

of

Schloss-berg et al. (25), our data show that different

viruses have different

chromatographic

pat-terns. However, these findings must still be

interpreted with care, since we have not col-lectedalarge enough series of samples of each virus to be assured of patternreproducibility. Further, we need to study specimens from

pa-tients with CNS neoplasia and demyelinating

disorders. The patterns associated with the clustered aseptic meningitis and the herpes zos-ter-related encephalitis, however, suggest that these patterns may indeed be specific for

cer-tain viruses. Iffurther analyses of

well-docu-mentedspecimens continue to differentiate

sys-tematically between CNS infections produced by different viruses, then GLC will be a very

useful clinical and epidemiological tool,

espe-ciallywhencontrastedwiththe poor yield from culture attempts and the time and effort re-quired to obtainseroconversiondata.

The problems of conventional diagnostic

techniques,combined withgeographically

scat-teredincidenceof viral CNSinfectionsandthe

inadequacy of sample size for analysis, have

been the majorfactors preventing conclusions

onthevalidityofthe GLCtechniquefor differ-entiatingbetweenviraletiologiesofCNS infec-tions. Wehope the data presented will

encour-age other investigators to providedocumented

2-ml specimens for further study of this

tech-nique.

Aside from the importance of adequate

sam-ple size, otherequipment, reagent, and

meth-odological details associated with this study

should benoted.Theextraction and

derivatiza-tion procedures are not complex, but they do

requirepracticetoachieve reproducibility. The

quality of reagentsused inthe analytical

proce-dureiscritical because of the extreme

sensitiv-ityof theECdetector,andsubstitutionsarenot

recommended. A temperature-programmable

gas chromatograph equipped with 24-foot (ca.

7.32-m) columns must be used to obtain the

necessary resolution ofthesecomplexmixtures.

Finally, inourexperience,neither the thermal

conductivitynorthe flameionizationdetectoris

selectiveor sensitiveenough to use inthis

ap-plication.

The datapresentedsuggestthat EC GLCcan be used to differentiate between tuberculous, cryptococcal,

viral,

and parasitic infections of the CNS. These differences are a result of the

presence ofcompounds that disappear as

pa-tients recover and are not present in normal

CSF. BycombiningGLC withmass

spectrome-try, it may bepossibleto

identify

someof these

compoundsandthusgainfurther

insights

into

thepathophysiology of CNS infections. If these compounds are toxic, new

therapeutic

ap-proaches to treating patients with meningitis

may also bepossible.

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32 CRAVEN ET AL.

ACKNOWLEDGMENTS

We thank John Bennett of the National Institutes of Health forthe assistance of the University Collaborative Study ofCryptococcal Meningitis in securing specimens and Amy Stineand Leo Kaufman of the CDC forverifyingthese specimens. We also thank Sharon Blumer of CDC for pro-viding the serogrouped specimens of C. neoformans and Michael Hattwick and colleagues for securing a wide vari-ety ofwell-documented viral specimens.

Thisresearch was supported by Naval Medical Research and Development Work UnitMR000.01.01.3121.

LITERATURE CITED

1. Alley, C. C., J. B. Brooks, and G. Choudhary. 1976. Electron capturegas-liquid chromatography of short chainacids as their2,2,2-trichloroethyl esters. Anal. Chem.48:387-390.

2. Amundson, S., A. I. Braude, andC. E. Davis. 1974. Rapid diagnosis of infection bygas-liquid chromatog-raphy: analysis of sugarsinnormaland infected cere-brospinalfluid. Appl. Microbiol. 28:298-302. 3. Brooks, J. B., C. C.Alley, and R. Jones. 1972. Reaction

ofnitrosamines withfluorinatedanhydrides and pyr-idine to form electron capturing derivatives. Anal. Chem. 44:1881-1884.

4. Brooks, J. B., C. C. Alley, and J. A. Liddle. 1974. Simultaneous esterification ofcarboxylandhydroxyl groups with alcohols andheptafluorobutyric anhy-dride for analysis by gas chromatography. Anal. Chem. 46:1930-1934.

5. Brooks, J. B.,C. C. Alley, J. W. Weaver, V. E. Green, and A. M. Harkness.1973.Practical methods for deri-vatizingand analysing bacterial metabolites with a modified automatic injector and gas chromatograph. Anal. Chem.45:2083-2087.

6. Brooks, J. B., W. B.Cherry, L. Thacker, and C. C. Alley. 1972. Analysis by gas chromatography of amines and nitrosamines produced in vivo and in vitrobyProteus mirabilis. J. Infect. Dis. 126:143-153. 6a.Brooks, J. B., G. Choudhary, R. B. Craven, D. Edman, C. C. Alley, and J. A. Liddle. 1977. Electron capture gas chromatography detection and mass spectrum identification of 3-(2'-ketohexyl)indoline in spinal fluids of patients with tuberculous meningitis. J. Clin.Microbiol. 5:625-628.

7. Brooks, J. B., D. S.Kellogg, C. C. Alley, H. B. Short, H. H.Handsfield, and B. Huff. 1974. Gas chromatog-raphy as a potential meansofdiagnosing arthritis.I. Differentiation between staphylococcal, streptococ-cal, gonococstreptococ-cal, and traumatic arthritis. J. Infect. Dis.129:660-668.

8. Brooks, J. B., D. S. Kellogg, L. Thacker, and E. M. Turner. 1971. Analysis by gas chromatography of fattyacids foundinwhole cultural extracts of Neis-seriaspecies.Can. J. Microbiol. 17:531-543. 9. Brooks, J. B., D. S. Kellogg, L. Thacker, and E. M.

Turner. 1972. Analysis by gas chromatography of hydroxy acids produced by several species of

Neis-seria. Can. J. Microbiol. 18:157-168.

10. Brooks, J. B., and W. E. C. Moore. 1969.Gas chromato-graphic analysis of aminesand othercompounds pro-duced by several species of Clostridium. Can.J. Mi-crobiol. 15:1433-1447.

11. Brooks, J. B., C. W. Moss, and V. R. Dowell. 1969. Differentiations between Clostridium sordellii and Clostridium bifermentans bygaschromatography.J. Bacteriol. 100:528-530.

12. Brooks, J. B., R. E.Weaver, H. W.Tatum,and S.A. Billingsley. 1972. Differentiation between Pseudomo-nas testosteroniand P. acidovoransby gas chromatog-raphy.Can. J. Microbiol. 18:1477-1482.

13. Cherry, W.B.,and C.W.Moss. 1969.Theroleof gas chromatography intheclinicalmicrobiology labora-tory. (Editorial)J. Infect. Dis. 119:658-662. 14. Greenlee,J. E., W. D. Johnson,Jr., J. F. Campa, L. S.

Adelman, and M.A.Sande.1975.Adult toxoplasmo-sispresentingaspolymyositis and cerebellar ataxia. Ann.Intern. Med. 82:367-371.

15. Maggs, R. J., P. L. Jones, A. J. Davies, and J. E. Lovelock.1971.Theelectron capture detector-anew mode of operation. Anal. Chem. 43:1966-1971. 16. Mitruka, B. M., and M. Alexander. 1967.

Ultrasensi-tivedetection of certain microbial metabolitesbygas chromatography. Anal. Biochem. 20:548-550. 17. Mitruka, B.M., L. E.Carmichael, and M.Alexander.

1969.Gaschromatographicdetection ofanvitroand in vivoactivities of certaincanineviruses. J.Infect. Dis. 119:625-634.

18. Morse, C. D., J. B. Brooks, and D. S. Kellogg. 1976. Electron capture gas chromatographic detection of acetylmethylcarbinol produced by Neisseria gonor-rhea. J. Clin.Microbiol. 3:34-41.

19. Moss, C.W., and W.B.Cherry.1968.Characterization of the C15 branched-chainfattyacidsof Corynebacte-rium acnes by gas chromatography. J. Bacteriol. 95:241-242.

20. Moss,C. W., and C. M.Kaltenbach. 1974.Productions ofglutaricacid:auseful criterion fordifferentiating Pseudomonas diminuta from Pseudomonas vesicu-lare.Appl. Microbiol.27:437-439.

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