Copyright ©1976 American Society forMicrobiology Printed in U.S.A.
Recent Experience with the Complement
Fixation Test
in
the
Laboratory
Diagnosis of
Rickettsial
Diseases
in
the
United States
C. C. SHEPARD,* M. A. REDUS, T. TZIANABOS, AND D. T. WARFIELD
CenterforDiseaseControl, Atlanta, Georgia30333
Received for publication 7 June 1976
Sera from patients suspected of having rickettsial infectionsweretestedinthe
complement fixationtestwith antigens preparedfrom the rickettsiae of Rocky
Mountain spotted fever (SF), rickettsial pox (RP), murine typhus, epidemic
typhus,andfromRickettsia canada (RC). Eight units ofantigenwereusedinall
casesandtwo units inman. Onlythosepatientswith antibodytitersof1:16or
higherwere included inthe study. Largely onthe basis of comparative titers,
thepatientsweredividedintotwogroups: 102withSF and35with infections by
oneofthemembers of the typhusgroup.The antibody titerswerehigher with SF
antigen than RP antigen in 72%of theSF patients, and in onlytwoSFpatients
was the RP titer higher, and then by only onetube (twofold dilution). There
seemed littleadvantageinincluding the RPantigen inthebatteryof rickettsial
antigens.Cross-reaction withatleastoneof thetyphusantigenswasobservedin
the serafrom64%of the SF patients. Itwas extensiveenough tobe confusing
(withinonetube) in17%witheightunitsof antigen, but the differentiationwas
moredistinct withtwounitsof antigen. The cross-reaction with typhus antigens
was asfrequentinchildrenwithSFasitwasinadults; thus, it isunlikelythat
these cross-reactionsresulted fromprevioustyphusvaccination.Theserological
differentiation between murine typhus and epidemic typhus was frequently
difficult, but the epidemiological background was distinct. Five patients had
higher titers to RC antigen, and four of these may possibly have had RC
infections.
Accurate diagnosis is basic to the control and
treatmentofaninfectiousdisease. The
labora-torydiagnosis of rickettsial diseases is
accom-plished chiefly through serological tests;inthis countrythe main serological method in use is
the complement fixation (CF)test with rickett-sial antigens. With good antigens, the test is
highly specific. Itis apparentlysensitive also,
butthisaspectof thetest (the abilitytodetect
allcasesofRockyMountainspotted fever[SF])
hasnotbeen rigorously investigated with any
serologicalprocedure.
SF isthe most important rickettsial disease
in the United States. Since the early 1960s, when the annual number ofcases
reported
wasabout 200, the number hasincreased
steadily.
Morethan800 cases were
reported
in1975.Thecase-fatality ratiohas decreased from the 20%
experiencedbefore thetetracyclines and
chlor-amphenicol were
available,
but it remains atabout7 to 8%.BecauseRickettsia rickettsii has
been difficulttogrowin
yolk
sacs,thediagnos-tic antigens forthe CF test had
usually
beenpreparedfrom the
etiological
agentofrickett-sial pox (RP), Rickettsia akari, which is an-other member of the spotted fever group and
one that grows more readily inyolk sacs. RP
antigens have seemed more useful than the available SF antigens (6).
Recently, however, methods for growingR.
rickettsii have improved considerably, so we
wished to compare the new SF antigens with
the RP antigens. Another question that has
arisen lately is the possible role of Rickettsia
canada (RC) inproducing adisease similarto
SF (2).If we were toinvestigatethis
problem by
comparinghomologous antibodytiters indiag-nostic sera, wewould needto useSFantigens,
rather thanRPantigens, forcomparison.
MATERIALS AND METHODS
TheLeprosy and Rickettsia Branch of the Center
forDiseaseControl(CDC)acts asthe reference
rick-ettsial diagnostic laboratory for the States. Some states send alltheirspecimens toCDC fortesting; some perform theirown tests and send only
occa-sionalsera toCDC.Theserumspecimenssubmitted
aresupposedtobeaccompanied byacompletedform
277
on February 7, 2020 by guest
http://jcm.asm.org/
278 SHEPARD ET AL.
that provides the standard clinical and epidemiolog-ical data, but frequently the information is incom-plete.
The rickettsiae were grown in the yolk sacs of hens'embryonated eggs. The strains usedwere:R. rickettsii,SheilaSmith; R.akari,Hartford;R.
moos-eri (R. typhi), Wilmington; R.prowazekii, Breinl;
and R. canada, McKiel. For all the rickettsiae ex-cept R. rickettsii, 7-day-old eggs were inoculated with a dilutionof infectedyolksac(usually1:100to 1:1,000)thatcaused the death of aboutathird of the embryos in6 to 7days. At this timeyolksacsfrom dead and living embryoswereharvested, aportion wassmeared and stained by the Gimenez method (4), andyolk sacs withgradings of4+ (100or more rickettsiae per microscope field) were selected for preparationof CFantigens.
R. rickettsii was grownby a modification of the Bell and Stoenner method (1)(personal communica-tionfrom Bozeman,Food andDrugAdministration, Bethesda, Md.). Yolk sacs of4.5-day-old embryos were inoculated with a dilution (usually 1:25) of infected yolk sacs that would kill almost all the embryosin 4to4.5days.Afterinoculation,the eggs wereincubated at35°C until death of theembryo; they werethen keptat33°C for another 48 h. For preparationof antigens,yolksacswithgradingsof 4+ were often selected; however, more than 95% wereusually satisfactory, sosometimesall the
em-bryoswereincluded.
Antigens were prepared by a modification of method 2 (7). Yolk sacs were ground in a tissue blenderto a 20%(wt/vol) suspensionin phosphate-buffered saline (pH 7.0) containing 0.1% formalin
andkeptovernightat4°C. As C. L.Wisseman, Jr.,
UniversityofMarylandMedicalSchool, Baltimore, Md.,suggested, the suspensionwasthen mixed with anequalvolume of 50% (wt/wt)sucroseand
centri-fuged
a(
20,000 x g for 1 h. The supernatant andlipid pellicle were discarded, and the walls of the tube werewiped clean. The sedimentwasthen
re-suspendedin 1 mlofphosphate-bufferedsaline for
each gramoforiginalyolksac, and thesuspension was shaken in a separatory funnel with about 3 volumes of anesthetic ether and allowed to stand
overnight. The next day the aqueous phase was
drawn off and thedissolved etherwasremoved by
bubblingwith nitrogen gas. Theresultantproduct
was used as the CF antigen. The antigens were checked in the CF test, and acceptable ones were dispensedin2-mlvolumesand frozenat-60°C.One vialwasthen thawedatatimeandpreservedat4°C untildepleted.
Antigens were titrated in the CF test (BCF, refer-ence 3) against late convalescent guinea pig sera (taken 2 months after inoculation) (6). The sera wereselected as much as possible for their ability to give a "square block" in a box titration, since an antigen shows verysimilar titers with all such sera. The inoculum for the guinea pigs was a suspension ofinfectious guinea pig blood and spleen, except in the case of RC, where it was an infected yolk sac diluted 1:1,000. SF and RP antigens were titrated against SF sera; murine typhus (MT), epidemic typhus (ET), and RC antigens were titrated against
homologous sera. Antigen titers varied from 1:8 through 1:64. Eight CF units were needed in the test (6), and acceptable antigenshad tobe free of anticomplementary activity at this concentration. Most lots ofantigen were acceptable.
All sera weretested against RP, SF, MT, ET,and RC normal yolk sac antigens and diluent (serum control). Thedilutionstestedwere1:8through1:256 in all instances; higher dilutions were sometimes tested to determine the end point. Only those sera free of anticomplementary activity and showing pos-itive (3 to 4+ fixation) in adilution of 1:16 are in-cluded here. The results with many non-rickettsial serashowthatfalse positives occur infrequentlyat serumdilutionsof 1:8 and veryrarely indeed at 1:16; the experience of many years with RP and other rickettsial antigens prepared by method 2 was the same. In the present work a retrospective study of serasubmitted in fiscal year 1972 andmostoffiscal year 1973wascarriedoutwithonebatch of antigens inconcentrations of 2 and 8 units, and all end points weredetermined (43 positive sera). Next, sera were tested every week or two as they came in routinely from May 1973 to November 1974 against 8 units of antigen, with repeats with 2 units of antigen and serumdilutions greater than 1:256 part of the time. Sera from patients with probable Q fever and Afri-cantick typhusareexcluded from this report.
Frequently, more than one serum sample was available from a patient, so one sample was
de-signed as the comparison serum. The comparison
serum wastakenasthe earliestonewiththehighest
homologous titer, and the results with this serum are used in most of the tables. With the following exceptions, the antigen producing thehighesttiter
wasdesignated as thehomologous antigen; results
with 2units of antigen were used for this purpose whentheywereavailable. The 102 SF patients in-cluded the three who hadequaltiterstoSFandMT;
the geographicandseasonal origins of these three
patients indicated that the assignment to SF was
correct. The23MTpatients includedeight who had
equaltiterstoET, RC,orboth (in three patients the
end point wasnot reached); the geographic origin indicated the MT assignment was correct. The five ET patients included one who had equal titers (without the end point being reached) to ET, MT, and RC; this patientwas in a typhus outbreak in Guatemala. The five RC patients will be discussed below. The assignments to SF or to the typhus group arethought tobe correct, but the assignments
be-tweenMT, ET,and RC represent our best judgment
(seebelow).
Inthe restof the text and in the tables the titers areexpressed as the serum end point dilution factor.
RESULTS
The geographicoriginof the sera is given in
Table 1. The geographic distribution of the SF
and MT patients reflects the endemicity of
thesediseases, inspite ofthefactthatmanyof
the states withthe mostSFperform their own
rickettsialserologicaltesting. The month of
on February 7, 2020 by guest
http://jcm.asm.org/
setwastaken from the form accompanying the sera or estimated from the date ofthe speci-mens. Asexpected, the seasonalincidence of SF ismuch more distinct than that ofMT. Thedip
inJuly has beenobservedinthe 1975 SFseason
also. Also as expected, the SF patients were
predominantly children, whereas the MT pa-tients were nearly alladults.
Only 9.5% of the patients had maximaltiters
of 16 or less (Table 2), and 50% had titers of greaterthan 256.
For the SF patients, the titers with SF and RPantigens arecomparedinTable3.
Only
twopatientshadhigher titers withRPantigen, and the accessory data confirmed that the disease
TABLE 1. Geographic origin, month of onset, and ageofpatients (when given)
Characteristic SF MT ET RC Total Geographic
New England 1 3 4
MiddleAtlantic 3 1 4
East North Central 4 2 6
WestNorthCentral 14 2 1 17
South Atlantic 29 2 1 1 33
East South Central 33 2 35
WestSouth Central 14 7 21
Mountain 3 1 4
Pacific 6 3 9
Foreign 1 2 3
Month ofonset
January 1 1 2
February 3 3 6
March 1 2 3
April 7 2 9
May 16 3 2 1 22
June 25 4 1 30
July 12 1 13
August 22 5 2 29
September 11 2 13
October 1 1
November 1 1
December 0
Age of patient
0-9 30 0 0 0 30
10-19 13 1 0 0 14
20+ 20 13 5 4 42
was, in
fact,
SF.Thetiter with SF antigenwasgreaterin72.2%of thepatients andmorethan
onetube
higher
in43.1%.Thus,
therewaslittleadvantage
inincluding
the RP antigen. (Sub-stantiated casesof RPhaveapparentlynotoc-curred for many years in the United
States.)
With15SF patients (15%),theserological diag-nosis would have been changed if SF antigen had notbeen included. In seven patients, noserological diagnosis could have been made(all titerss8), andineight patients, the
serological
diagnosis would have been MT (fourpatients), ET (twopatients), orRC (twopatients) (sincethe titers with one or more of these antigens
werehigher than thatwithRP).
The degree of cross-reactivity with SF and typhusgroup (T) antigensisshowninTable4. Frequently, when the cross-reactionwas
exten-sive with 8 units ofantigen, itwas much less
with 2 units. One might expectthatthe SF-T
cross-reaction would bemore easily detectable withhighserumtiters,onthe assumptionthat
thecross-reactingantibodiesareamore-or-less
constant fraction ofthe total. The results in
Table5, however, show thatsuchatendencyis
not strong and that many sera with titers
greaterthan256didnot cross-react.
Cross-reac-tionswere morefrequentwith SF sera (65 out
of 102 = 64%)thanwith Tsera (10out of 36 =
28%).
The positive results with T antigens in SF
are sometimes attributed to previous
vaccina-tionwithETvaccine (2), since inthe pastthis
vaccinehas beengivento mostofthemembers
of the armed forces. The results in Table 6, however, show that typhus vaccination could
have had littleeffect onourresults. Reactions
withoneof the Tantigenswereasfrequentand
asextensivein childrenastheywereinadults.
The patients withhighertiterstooneofthe
typhus antigens weredivided into MT, ET, or
RC for the tables, primarily on the basis of
comparative titers andsecondarilyonthe basis
ofaccessorydata.Thus, asshown inTable7, of
the30patientswithhighesttiterstoMTorET,
8 had equal titers with 8 units of MT and ET
TABLE 2. Highestserumtiters observedforeachpatient(8 unitsof antigen)
Serologicaldiagno- No.ofpatientswithindicatedserumtitera
sis 8 16 32 64 128 256 >256 512 1,024 2,048
SF lb 5 12 13 17 6 29 11 8 0
T(all) lb 4 2 2 3 3 7 6 6 1
1.5%c 8.0% 18.2% 29.2% 43.8% 50.4% 76.6% 89.1% 99.3% 100.0%
aTitersareexpressedas serumendpointdilutionfactorinthis andother tables.
IThetiter was 16with2 unitsofantigen.
c Cumulativepercent.
4,
on February 7, 2020 by guest
http://jcm.asm.org/
TABLE 3. Patientswith SFresponses: comparisonsof titers to SF and RP antigens
Patients with SFresponses
-la 0 1 >1 2 3 >3 4 5 6 >6 7
2 22 25 5 13 6 3 3 2 1 3 1 16b
2.3% 25.6% 29.1% 5.8% 15.1% 7.0% 3.5% 3.5% 2.3% 1.2% 3.5% 1.2% 43.1%
aNumberoftubes(twofolddilutions) withSF titer higher than RP titer.
bComparisonnotpossible becausetheend pointswere notreachedwitheither SForRPantigens.
TABLE 4. Comparativeserum titers to SFandT"antigens
SFpatients (102)
ob 1 2 3 4 5 6 7 8 >1to >5 >6
7C 10 11 17 7 9 9 2 1 17 11
6.9%d 16.7% 27.5% 44.1% 51.0% 59.8% 68.5% 70.6% 71.6%
T(MT, ET,orRC)patients (35)
o0 -1 -2 -3 -4 -5 -6 -7 -8 >-6
-1' 2 6 4 1 3 4 5 2 6 1
2.9%d 85% 25.7% 37.1% 40.0% 48.6% 60.0% 74.3% 80.0%
aMT, ET, or RC.
Number of tubes (twofold dilutions) withSF titers higher thanhighestT.
Fourweretestedwith 2 units ofantigen also, and the SF titer was found to be 1, 4, 6, and 6 tubeshigher, respectively.
*tCumulative.
Thisserum wastestedwith 2units of antigen also, and the SF titer was found to be 5 tubes lower.
TABLE 5. Proportion of SF patients withoutcross-reaction withTantigensandproportionof Tpatients
without cross-reaction to SFantigens
Serological No.withoutcross-reaction/no. positivewithhomologoustiter
diagnosis 8a 16 32 64 128 256 >256 512 1,024 2,048 All
SF 0/1 4/5 6/12 4/13 7/17 3/6 11/29 2/11 0/8 37/102 = 36%
MT 1/1 1/1 1/1 1/2 3/3 2/3 4/6 1/1 2/5 0/1 16/24 67%
ET 2/2 1/1 1/1 2/3 0/1 6/8 = 75%
RC 1/1 1/1 2/2 4/4= 100%
a Homologous titer(8 units of antigen).
antigens. Seven ofthese patients lived in the
United States, and their exposure occurred in
areaswhereratsareknown to be infected with
MT; one patient was from Colombia, and his
titers with 2 units of antigen were one tube
higher withMT. Oneoftheeight patientslived
in Hawaii and probably had MT, but with 2
units of antigen her titers were distinctly
higher with ET antigen. Another patient living
inCalifornia hadaone-tube highertiter toET
antigen, butprobably had MT. Withthese
ex-ceptions, the classification asMTorET inthe
tablesisprobablycorrect;all patientsclassified
as MT lived in or acquired their infections in
MT-endemicareas,and all of those classifiedas
ETlivedinforeign areasknowntohave ETor
had been born many years before in eastern
Europe. All the patients classified as RC had
highest titers to this antigen and areconsidered
below.
The difficulty in differentiating MT and ET
patientsonthe basisof the CF resultsis
sum-marized in Table 7. Of the 30 MT and ET pa-tients, only 16 had differences of two tubes or
more; these resultswereobtained with 8 units
ofantigen. Thedifferentiation was sometimes
more distinct with 2 units of antigen; the
differ-ence was increased in six patients, but itwas
not in two.
A comparison of RC titers with MT or ET
titers is shown inTable 8. In 11 of the 102 SF
patients, the RC titers were higher than the
MT or ET titers; however, these are not of
concernbecause the SF titerswerehigherinall
cases,and the correctdiagnosiswasverylikely
SF. The confusing outcome that would have
280
SHEPARD ET AL.on February 7, 2020 by guest
http://jcm.asm.org/
CF TEST DIAGNOSIS OF 281
resulted if the SF antigens had not beenused
has beenpreviously discussed (Table5).
Thepatientswhose highesttitersweretoRC
are shown inTable 9. The fourth patient had
had typhus in India many years ago and was
experiencing repeatedfebrileepisodes; the low
titer, 16, and the atypicalclinical picture
sug-gestthat thecurrentillnesswasnotrickettsial
TABLE 6. SFpatients-amountofcross-reactionwith
anytyphus antigen; results with 8 units of antigen
SF patients
Amt of cross Age (yr) Sexa Total One tube
None Oetborless
0-9 M 22 11 3
F 8 3 2
M&F 30 14 5
10-19 M 9 5 0
F 4 2 0
M&F 13 7 0
20-69 M 10 4 1
F 10 5 2
M&F 20 9 3
a M, Male; F, Female.
in origin.Thefifth patientworkedinabuilding
in which a rickettsial laboratory was located,
butonanother floor, and had hadsome
recrea-tionalexposuretoticks. The first three patients
werefemales in northern states, and their
dis-eases were severeand began inwinter. These sera werepart of the batch studied
retrospec-tively; we didnot observe similarpositives in
the next three winters (1973 to 1974, 1974 to
1975, 1975to 1976).
TABLE 7. Comparative titerstoMTand ETantigens
(8 units)
Serological Titers
diagnosis _2a -1 0 1 2 3 4 >1 >5
-ET 1 4 lb 1
MT 7 2c 6 1 1 2 1 3
RC 5
aNumber of tubes with MT titers higherthan ET.
bThis patient, a 13-year-old female livingin Ha-waii, had titers of 512 with8units of MT, ET, and RC antigens and 8, 128, and 8 with2 units ofthe three respective antigens.
cOne of these patients, a44-year-old male living inCalifornia, had titers of 16, 32, and <8 with8units ofMT, ET, and RC antigens, respectively.
TABLE 8. Comparative titers, RC versus MT or ET (8 units of antigen)
Titers Serological
diag-nosiea -5b -4 -3 -2 <-1 -1 0 1 >1 2 >2 3
>5
doneSF (102) 2 3 3 17 67 5 5 1
MT(23) 1 1 1 3 1 9 5 2
ET(7) 1 2 1 1
RC (5) 2c 2 1
a Number of patientsisshowninparentheses.
bNumber of tubes that RC titerwasgreaterthan MTorET(higher of the two)titer.
c When the seraweretested with 2 units of antigen, the RC titers were 1 and 2 tubes higher, respectively.
TABLE 9. Patientswhosehighesttiterswere toRC
Dateof Time after Units of Titer Clinicalimpression CDCno. Sex Age State onset onset antigen SF RP MT ET RC andremarks 73046911 F 36 KA 1/10/73 1 <2months 2 <8 <8 32 64 128 SF and MT
8 <8 <8 256 256 512
73048951 F 14 VT 12/1/72 1 <3months 2 16 64 128 64 512 FUObpossible 8 256 256 1,024 1,024 1,024 subacute
2 <3months 2 64 <8 256 32 1,024 bacterial
8 128 128 512 512 512 endocarditis 3 <3months 2 <8 <8 8 <8 32
8 <8 <8 32 128 128
73051271 F - MA 2/73? 1 <1month 2 <8 <8 64 32 128 MT
8 <8 <8 512 512 512
74058398 M 44 NH ? 1 Many 8 <8 <8 8 8 16 Recurrenttyphus?
years Has hadtyphus.
74059986 F 49 MD 5/1/73 1 9months 8 <8 <8 64 64 >256 MT?
aSerum number.
°FUO,Feverofundeterminedorigin. VOL. 4, 1976
on February 7, 2020 by guest
http://jcm.asm.org/
DISCUSSION
SF is the mostimportant rickettsial disease
in theUnitedStates; thus, it ishighlydesirable
that we be able toprovide accurate laboratory
diagnosis for this disease. The CF test with
rickettsial antigens seems to provide the
needed specificity to differentiate it from MT,
the other important cause of the typhus
syn-dromeintheUnited States. Theothermembers ofthe spotted fever group are easily
differen-tiated on clinical or epidemiological grounds.
RP is a mild disease with a vesicular rash,
sometimes confused with chicken pox; to our
knowledgethedisease hasnotbeenseen inthe
United States for about 20years. Recently we
have received sera froma dozen or so casesof
African tick typhus each year, all with a
his-tory ofexposure in Africa.
The results showed little advantage in
in-cluding RPantigens among those used to test
diagnosticsera. RP antigensneedtobekepton
hand for particular specimens, however, at
least in this laboratory.
Crossing with MT antigenswasseen in 64%
of SF sera, butthedegree ofcrossingwas
usu-ally notbothersome, especially with2 units of
antigen. In only 28% ofMT and ET sera was
there cross-reaction with SF antigen.
Conse-quently when cross-reaction between SF and
one ofthe T antigens is seen with 8 units of
antigen, the test needs to be repeated with 2
units.
The serological differentiation of MTand ET
is difficult because the two rickettsiae are
closely related antigenically; moreover,the dif-ferentiation is unreliable because, after
expo-sure to ETantigen, the serological response to
MTinfectionisfrequentlyET in
specificity
(5).In the patients studied here, two of the seven
with highest titers to ET antigen were very probably MT in etiology.
There are several technical difficulties in the
serologicaldiagnosis of RC infections. Only one
strain of RC has been isolated, so there is no
way of knowing whether the strain used is
antigenicallyrepresentative. Another difficulty
is encountered in determining the dilution of
antigen to beused. For antigen titrations of the
otherrickettsiae, wehave used sera ofguinea
pigsinfected withguineapigpassage material;
these guinea pigs have had typical
experimen-tal disease. RC infections are only subclinical,
however, so theguineapigs were infected with
yolk sac material and bled after an arbitrary
time.
The technical differences between our
meth-ods and those of Bozeman et al. (2) do not
appear to be substantial. The (soluble type)
antigens were prepared similarly, that is, by
centrifugal sedimentation of the rickettsiae,
followedby resuspensioninbuffered saline and
ether extraction. In our case, the sedimentation
tookplacein 25%sucrose.Guineapigserawere
used tostandardize the antigens, theirs taken
21 to 28 days and ours taken 60 days after
inoculation. They used 4 to 8 units ofantigen,
and we used 8 units (in the initial screening).
They used a typhus groupantigen consisting of
a mixture of equal parts of murine and
epi-demic antigen, whereas we used the separate antigens. Our results would need to be com-pared with those they obtained with typhus
group and K. canada antigens.Althoughinthe
table the latter is placed under the column heading "Specific" antigens, the text states that "the results presented were obtained with
solu-ble type antigen."
The principle difference in the results of the
two studiesis that theirpatients had little or no
crossing with antigens of the typhus or SF
group,whereas all of ours had extensive
cross-ing. They state, however, that among 60 SF
patients, 18 had antibodies that reacted with T antigens; however, only 3 of the 18 had
antibod-ies that reacted with RC antigenand then to
about the same titer. All of their RC patients
hadexposure to ticks.
Of the five patients we encountered with
higher titers to RC, one had only a low titer thatprobablyreflected atyphusinfection many
years before. Of the other fourpatients, three
were infemales in northern states in the winter
time, who seemedunlikely, therefore, tohave had SF or MT. These three were found in the
retrospective survey, andnosimilar cases have
beenencounteredsince.
Bozeman et al. (2) attributed the typhus
cross-reaction in SF cases toprevious
vaccina-tionwith ETantigen. Ourdata(Table6),
how-ever, showed that cross-reaction was as
fre-quent in persons who were unlikely to have
been vaccinated.
Thechiefdisadvantage of theCF testforthe
diagnosis of rickettsial diseases is, of
course,
the slowness with which antibodies appear.
Al-thoughCFantibodiesaresometimes detectable
atthe end of the first week ofillness when 8
units ofantigen are used (6), antibody is
usu-ally not demonstrable until the end of the sec-ond week; indeed, it may not appear until the
fourth week. SF patients who are treated
promptly with tetracycline orchloramphenicol
arefrequently well and discharged from
medi-cal care before CF antibody appears. Other
testsmay be more satisfactory in detecting
im-munoglobulin M or other early antibody.
Dur-ing the 1975season acomparisonof theCF test
on February 7, 2020 by guest
http://jcm.asm.org/
CF TEST IN DIAGNOSIS OF RICKETTSIAL DISEASES 283
with the microagglutination and indirect flu-orescent antibody tests has been carried out in this laboratory. These two tests gave nonspe-cific reactions in lower titers with non-rickett-sial sera, however, and when comparisonswere made at the dilutions that were needed to pro-vide acceptable specificity for each test the CF test was at little disadvantage in detecting (specific) early antibody (unpublished data). Still other procedures for the demonstration of rickettsial antibody will undoubtedly soon be-comeavailable and should be compared system-atically.
Early diagnosis might be achieved rapidlyby detecting specific antigen, but techniques are not yet developed. R. rickettsii may beisolated from the blood of nearly all cases of SF in the first week of illness and before specific treat-ment has beenstarted, but the answers are not immediately available. In fatal cases the labo-ratory diagnosis may sometimes be madeonly
by
isolationprocedures. No laboratory diagnos-ticprocedure is now available that will provide a specific laboratory diagnosis in time to help the physician in his decision about therapy. The clinical picture of SF is usually distinct, withfever, headache, rash on the extremities, mental changes (such as dullness, stupor, or delirium), and a history of tick exposure.More-over hyponatremia is usual and
thrombocyto-penia isfrequent. In the most severe cases, in which death occurs at about the end of the first week, we have observed, however, that the ma-culopapular rash is often indistinct or absent and the clinical diagnosis is much more diffi-cult.
LITERATURE CITED
1. Bell,E. J.,and H.G. Stoenner. 1960. Immunological
relationships among the spotted fever group of rick-ettsias determined by toxin neutralization tests in mice withconvalescent animal serums. J. Immunol. 84:171-182.
2. Bozeman, F. M., B. L.Elisberg,J. W.Humphries,K.
Runcik, and D. B.Palmer,Jr. 1970. Serologic
evi-dence of Rickettsia canada of man. J. Infect. Dis.
121:367-371.
3. Casey, H. L. 1965.Adaptationof the LBCF methodto
microtechnique, p. 31-34. In Standardizeddiagnostic complement fixationmethod andadaptation tothe micro test. U.S. Public Health monograph no. 74.
U.S. GovernmentPrinting Office, Washington,D.C.
4. Gimenez,D. F. 1964. Staining rickettsiaeinyolk-sac
cultures. Stain Technol.39:135-140.
5. Goldwasser,R. A.,and C. C.Shepard. 1959.
Fluores-centantibodymethods in the differentiation of
mu-rine and epidemic typhus sera; specificity changes
resulting fromprevious immunization. J.Immunol.
82:373-380.
6. Hersey,D.F., M. F.Colvin,andC.C.Shepard.1957.
Studiesontheserological diagnosis ofmurinetyphus
andRockyMountainspotted fever.II.Human
infec-tions. J.Immunol.79:409-415.
7. Topping, N. H., and C.C.Shepard.1946.The prepara-tionof antigens fromyolksacsinfected with
rickett-siae. Public HealthRep.61:701-707.
VOL. 4, 1976
