Serologic Diagnostic Methods

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SEROLOGIC DIAGNOSTIC METHODS

Basis for Serologic Reactions:When certain substances are introduced into the animal

body the tissues respond with the formation of special gammaglobulins. These gamma globulins are called" antibodies"

and are capable of combining in a specific manner with the

substances called "antigens" that evoked their formation.

Complete antigens are usually proteins. The specific chem-

ical group in a complex antigen responsible for antibody

formation is called' 'hapten. "Antigen- antibody reactions are highly specific. An

antibody will react only with the species of antigen which

evoked its formation. This can be illustrated as the combin-

ation of a given chemical configuration with its complementary

image:

Serologic and immunologic methods utilize the specificity

of antigen-antibody reactions for the diagnosis of disease.

In particular, diseases due to infectious agents are character-

ized by the development of specific antibodies against the

responsible microorganism. The presence of a specific

antibody in the serum thus suggests past or present infectionof the person with the microorganism. Detection in the test

tube of specific antibodies is, therefore, a valuable tool in

the diagnosis of diseases suspected to be of infectious origin,

particularly if the etiologic agent cannot be recovered.

While antigen-antibody reactions "are specific, cross re-

actions will occur in serologic procedures and immunologic

tests between antigens possessing closely related chemical

groups and antibodies. Knowledge of this possibility will avoic.

confusion.

Time for Taking Specimens:For most conclusive results, always obtain one specimen

of blood serum as early as possible in the disease, another

about ten days later, and a third about four weeks after onset

of the illness.

er of Drawing Blood:

For proper performance of all serologic tests the blood

5-e...-.unmust be obtained properly. Venous blood is drawn

~aseptic precautions (see p. 132) and permitted to clot in~ 5:erile test tube. The clot is separated from the test tube

z : : . . :. and the serum left at refrigerator temperature overnight

.::;>erroIt clot retractIon. The tube is then centrifuged and

- c.ear serum, free from hemolysis, is transferred by pipet: : : > another lIghtly stoppered, sterile tube. The serum

~- -':dbe kept cold and brought to the laboratory as quickly as

- -s:ble.

= = - - :-pretation of Results:1: antibodies are present in the same concentration (titer)

~ a.:: :hree specimens obtained as described on p. 286, they

-e. ~ost certamly acquired in the past and have nothing to

.:n the present Illness. If, on the other hand, their con-

_-:::-a"on (titer) rises very markedly in the course ofthe

;.....::.ESS (L e., from the first to the third specimens) they help

es:a.blish the definite diagnosis.

:-x::-_-\ntitoxin Neutralization:

er-ain bacteria, e. g., Corynebacterium diphtheriae or

s=-:dium botulinum, produce soluble toxins. Antibodies to

- _~e: xins, i. e., antitoXins, are able to neutralize them

-- =:xed in proper proportions. This reaction takes place

~~. a.Dsence of complement (see p. 299). This principle is

- -_ :n the typIng ofbotulinus toxin and in the virulence test

_ ::"?!:ilieria bacilli.

- _::::::a 'on Tests:

-e:-..ain organisms evoke the production of antibodies in

-- and these antibodies can agglutinate the causative

~m5 in vitro.

--~cations:

.c.entification of isolated bacteria by slide or tube test

see p. 279) by mixing with known sera.

easurement of antibody concentration in patient's

senun by mixing serum dilutions with diagnostic anti-

gens, especially the following:

li. almonella species (typhoid, paratyphoid fevers,

etc. )Brucella (undulant fever)

c. Proteus OX19, OX2, OKX(rickettsial diseases;

see p. 295)c. Pasteurella pestis (plague)e. Pasteurella tularensis (tularemia)

Leptospira species (leptospirosis)


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3. Other diseases - In some cases of "primary atypical

pneumonia, " a nonspecific agglutination reaction with

streptococcus MGis positive (see p. 297); cold agglu-

tination tests are also frequently positive (see p. 293).

Sera from patients with rheumatoid arthritis often

agglutinate sensitized sheep cells (see p. 294).

B. Technic of Agglutination Tests:

1. Tube method - Make serial twofold dilutions of serum

with saline in the following manner. Place O . 5 ml.

saline in each of a series of small test tubes. Add

O . 5 ml. serum to first tube, mix well by drawing up

and expelling repeatedly from pipet, and transfer 0.5

ml. to second tube. Proceed similarly until last tube

of the row is reached, then discard 0.5 ml. To each

tube add 0.5 ml. ofa bacterial suspension suitably

standardized for density. Shake the rack of tubes,

then incubate overnight at 37QC. (98. 6QF.) or 50QC.

(122QF. ).

A positive reaction (++++) consists in complete

clumping of the bacteria with complete clearing of the

supernatant fluid. A negative reaction (-) consists ofuniform turbidity in the suspension without clumped

sediment.

2. Slide agglutinations are often useful for rapid identi-

fication ofbacteria. A suspension of unknown organ-

isms from a fresh culture is mixed with a drop of

specific antiserum on a glass slide. On another area

of the same slide the organisms are mixed with a drop

of saline. After standing or gentle rotation of the slide

for a few minutes, the slide is observed grossly and

under the microscope. A positive result is indicated

by rapid clumping ofbacteria and loss of motility in

the serum drop but not in the saline drop (high dryobjective, subdued light) (see p. 279).

A similar rapid slide agglutination test may be per-

formed with commercial salmonella or brucella anti-

gens for the identification ofantibodies in unknownsera.

C. Interpretation of Agglutination Tests for Salmonella Infec-

tions (Typhoid, Paratyphoid, Etc.):

1. "0"and "H" antigens and antibodies - Many motile

bacteria have two principal antigenic components:

a. The somatic "0"antigen (=Ohne Hauch) is obtain-

ed when thick suspensions of bacteria are treated

with an equal quantity of absolute alcohol with

vigorous stirring and shaking. Incubate the mixturefor 12 to 24 hours at 37QC. (98. 6QF.), then shake

well. Add a quantity of saline equal to the amount

of alcohol added, to reduce alcohol concentration

to 330/0,and store in refrigerator. Adequate"0"

antigens are obtained from E. coli by heating the

culture for one hour in boiling water bath.

b. The flagellar "H" antigen (=Hauch) is prepared by

growing the bacteria on agar in Blake bottles and

washing the growth off with saline containing O . 50/0

formalin. Shake well, and store in the refrigerator.

In"H" antigens the formalin treatment preserves

the flagellar structures on the surface while in'.'0"

antigens the treatment removes them. A third

group of somatic"K" antigens are thermolabile,and occur as envelopes surrounding the cell body.

They may interfere with"0"agglutination and must

sometimes (e. g., in E. coli serotypes) be destroyed

by heating before the"0"antigen can be determined.

One important "K" antigen is the Vi antigen ofviru-

lent typhoid bacilli.

2. Factors in interpretation - For the diagnosis of infec-

tion by serologic tests the following are important:

a. The best proof of active disease (e. g., typhoid)

other than identification ofthe organism is the find-

ing of a rising agglutination titer when serum is

secured from the patient at intervals during the

second and third weeks of the illness.

b. Significant agglutination titers may persist in the

blood for years after clinical or subclinical infec-

tion or after vaccination. The finding of a single

positive agglutination test therefore does not prove

the existence of active disease.

c. There are some cross-reactions between various

enteric organisms encountered in agglutination

ests. The organism agglutinated by the patient's

serum in the highest dilution is ordinarily con-

sidered the most significant.

c. The following antigenic patterns are often encoun-tered in salmonella infections:

1:80 or more, Low

not rising

< 1:40 1:40 or

more

and 3rd weeks

. ac ive typhoid

:erer

-=-cination in

:.:.e past

_:L~:er--:e

Titers observed with antigens

"0" "H" "Vi"

1:80 < 1:40 Lowand

risin

Similar" receptor analysis" can occasionally be

carried out in enteric fevers other than typhoid,

e. g., dysentery.

= - . _ - _ : : : ~ Tests:.--=:ibodies against soluble antigens (' 'precipitins' ') are

~onstrable by their formation of a visible precipitate


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as the result of antigen-antibody reaction. As the precipitate

is often soluble in excess of either antibody or antigen, the

two have to be mixed in accurate proportions. Precipitin

tests are used clinically for the diagnosis of syphilis (see p.

298), for the typing of ,B-hemolytic streptococci, for the

serologic identification ofproteins (e. g., identification of

blood stains), and in trich~nella and echinococcus infections.

Opsonocytophagic Tests:

Antibodies which enhance the ability ofleukocytes to in-

gest bacteria are called "opsonins." They can be demonstrated

by mixing patient's fresh citrated blood with a suspension of

bacteria and comparing the degree of phagocytosis with that

of controls. Such tests are occasionally employed in brucel-

losis but are of doubtful significance for diagnosis.

Bacteriolysin Test:

Antibodies can occasionally be demonstrated by their

ability to dissolve bacteria. While the antigen-antibody re-

actions described above take place in the absence of com-plement, the latter is an essent.al component in this and the

folloWing reactions. Complement is a substance present in

most fresh mammalian sera and destroyed by heating at 56C.

(138F. ) for 30 minutes. This procedure is called' 'inacti-

vation" of a serum. In most tests complement is used in the

form of pooled guinea pig serum of established satisfactory

potency (fresh or reconstituted from the dehydrated state).

In a test for bacteriOlysis, suspensions of the organism

are mixed with serum which is being tested for antibodies.

Complement is added and the mixture incubated at 37C.

(98.6 F.) for one hour, or (in the case of cholera) injected

intraperitoneally into guinea pigs. Control mixture s con-

taining normal serum are included in the test. At the end of

the incubation period the organisms in contact with specific

antibodies will have dissolved, while those mixed with normal

serum should be unchanged. The test is used mainly in chol-

era and leptospirosis (as agglutination-lysis).

Fluorescent Antibody Technics:

A new serologic technic is based on the conjugation of anti-

body molecules with fluorescent dyes (e. g., fluorescein,

rhodamine). Such" lab'eled" antibody may be used to locate

antigen microscopically because ofthe high specificity of the

antigen-antibody bond. Fluorescent-Iabeled antibody can

rapidly identifY specific microorganisms in smears fromcultures, pus, tissue, or exudates, and can establish the

localization of antigens in tissue section. This technic is

used for bacteriologic and virologic diagnosis and for the

study of"hypersensitivity" diseases.

This technic can also be used for indirect staining:

antigen + its antibody A + anti-A-antibody-fluorescent

e. g., + (e. g., antivirus + (e. g., antihuman globulin

'-;:rus) human antibody) from rabbit)

Such indirect technics permit the localization of some

a--"gens or antibodies which cannot otherwise be established

_:-..present methods.

-::::::::> ement Fixation Tests:

7he reaction consists oftwo parts:

....Part I - Mechanism of Reaction: Specific antibody com-

bines with ("fixes'~ complement only in the presence of

corresponding specific antigen:

:J-.?eC

iliC00Complementbound

--.. ("fixed")

~

,,-- ~:;c [] 0Complement_ not bound

(' 'not fixed")

:= . Part 2 - Test for Presence of Free (Not Bound) Com-:Lement: "Hemolytic system" is added to above mixture

. ~antigen + antibody + complement. 'Hemolytic system"

onsists of sheep cells + anti-Sheep cell hemolysin (i. e.,

serum from rabbits repeatedly injected with sheep cells).

-: free complement is present, it will combine with and

::emolyze the sheep cells (i. e., if the original antigen did

fit the antibody, the two did not combine, hence no

omplement was bound).

8U[]~LYSIS


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This complement fixation test is negative. Hemolysis

is present.If free complement is not present (because it was all

bound by original antigen-antibody combination), the

sheep cells will remain intact:

OuNo

complement

available _ NO

LYSIS

Sheep cells + Hemolysin

This complement fixation test is positive. No

hemolysis.Between parts 1 and 2, the antigen-antibody-com-

plement mixture is incubated for varying periods at 37C.

(98. 6F.) or kept at refrigerator temperature for

fixation" of complement prior to addition of the indicator"hemolytic" system.

C. Complement fixation tests are used commonly in the

diagnosis of the following diseases:

1. Syphilis (see p. 298).

2. Viral diseases (see p. 296).

3. Rickettsial diseases (see p. 295).

4. Fungous diseases (especially coccidioidomycosis,

histoplasmosis, blastomycosis).

They are occasionally employed in the diagnosis of

brucellosis, parasitic diseases (cysticercosis, trichin-

osis, echinococcosis) and gonorrheal arthritis.

Sheep Cell Agglutination Test for Infectious Mononucleosis

(Paul-Bunnell Test, "Heterophil Agglutination Test"):

This is a nonspecific reaction based on the finding that

persons with infectious mononucleosis develop a high titer of

sheep cell agglutinating antibodies.

A. Technic: Sheep blood is obtained fresh and the cells

washed three times with saline. A 2%suspension of cel

is then prepared. Sera from patients are inactivated at

56C. (133F. ) for 30 minutes. Whenever possible ser~

should be obtained as early as possible in the illness and

ten days later, to observe the rise in antibodies. A

series of small test tubes is set up as shown in chart on

next page (Davidsohn, JAMA 108:289, 1937).

B. Problems ofInterpretation:

1. Normal persons may have agglutination titers up to

1: 112.

2. After the injection of animal serum (e. g., antitoxin).

individuals often develop high titers of agglutinins

(which will agglutinate sheep cells).

3. The agglutinins mentioned under 1and 2 can be re'

moved from the serum by absorption with guinea pig

kidney.

4. The agglutinins developing in mononucleosis are not

absorbed with guinea pig kidney but are absorbed bywashed and boiled beef red cells.

.::be ~o. Saline Serum Sheep cells Final serumml. m!. m!. dilution (titer)

l 0.4 0.1 O . 1 1:7

2 0.25 0.25 of Tube 1 O . 1 1:14

3 0.25 0.25 of Tube 2 O . 1 1:28

4 0.25 0.25 of Tube 3 0.1 1:56

5 0.25 0.25 of Tube 4 O . 1 1:112

5 0.25 0.25 of Tube 5 0.1 1:225

. 0.25 0.25 of Tube 6 O . 1 1:450

3 0.25 0.25 of Tube 7 O . 1 1:900

9 0.25 0.25 of Tube 8 O . 1 1:1800

10 0.25 0.25 of Tube 9 0.1 1:3600

1: 0.25 0.25 of Tube 10 O . 1 1:7200

.2 0.25 - - - - - O . 1 Control

Keep tubes at room temperature for two hours or at

-C. (98. 6F.) for one hour, and place in refrigerator

.ernight. Shake gently and note clumping of cells.

'.' .' gg utination Test:

'- some cases of "primary atypical pneumonia" (PAP)

"'--ces appear in the serum which are capable of agglu-

--- -" human group 0red cells in the cold, but not at room--::.;ature or at 37C. (98.6 F.). The test is performed by

.- serial twofold dilutions ofthe patient's serum in saline

~ .. g an equal amount of a 1"/0suspension of washed

'. group 0red cells. The mixtures are shaken well and

- :he refrigerator overnight. A positive result is indi-

_~ .. :inding clumped cells immediately on removing the

.:;:;om the refrigerator which do not remain clumped after

~5 have been at room temperature for three to four

-.:;. Control tubes with normal serum and with saline-red

_ ::::ix1ures are included.

7-p degree of positivity of this test depends on the etiology

. some extent the severity of the illness. PAP caused by-~_ ses is never associated with a positive cold agglu-

_ - test. PAP due to the Eaton-Meiklejohn pneumonitis

--5 and perhaps other agents gives cold agglutination tests

- _ :x>rtion to the severity ofthe illness. Early treatment

::.cases with tetracycline drugs seems to interfere with

~ elopment of a positive test.


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Antistreptolysin Titer:

Persons infected with l3-hemolytic streptococci often

develop antibodies against the hemolysin 0 produced by the

streptococcus. This antibody can be tested for by its ability

to inhibit hemolysis of red cells by a standardized strepto-

coccus hemolysin. The test should be carried out only in a

laboratory having considerable experience and standardized

reagents at its disposal. A reliable technic is indicated by

Rantz, L. A., et al. , Am. J. Med. 5:3, 1948.Finding persistently high antistreptolysin titers (in excess

of 125 units) suggests recurrent or persistent infection with

hemolytic streptococci and reaction to such infection. NOTE:

Adequate penicillin therapy of hemolytic streptococcus infec-

tions often interferes with the development of antistreptolysin

in high titer.

Serologic Tests for Rheumatoid Arthritis:

Most patients with active rheumatoid arthritis possess an

unusual serum protein which is capable of reacting with gamma

globulin. This substance (' 'rheumatoid factor") can be demon-

strated by several serologic tests. Most of these tests con-

sist in the agglutination of particles (latex, bentonite, tannic-

acid-treated red blood cells, etc.) onto which human gamma

globulin has been adsorbed by diluted serum from rheumatoid

patients. The interaction of rheumatoid factor and gamma

globulin can also be demonstrated as a precipitin reaction and


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:" 01 1 1 ' III '" ,' I l :I t C l l l I I < A-_ .

Itu h - Dc ('UI'J'I~IH'( V(~l'tm W.


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Disease Etiologic Agent Specimen Submitted for Examination Test Remarks

Croup Hemadsorption, Throat swab; blood serum, Virus isolation, Different viruses in

(infantile) croup-assoc. acute and convalescent. hemagglutination- different years.

viruses inhibition.

Denflue Denllue virus Blood serum, acute and convalescent. * Virus neutralization. Not often available.Encephalitis Specific viruses Blood serum, acute and convalescent. * Virus neutralization, Only rise in titer diagnostic.

(Japanese B, St. Louis, complement fixation.

Western and Eastern

Equine, Venezuelan) Brain (autopsy). Virus isolation.

Gingivostomatitis, Herpes simplex Vesicle fluid, saliva, spinal fluid. t Virus isolation, In eggs, mlce, raoolts, tissue cuJ.-herpes labialis, cold virus Blood serum, acute and convalescent. * virus neutralization, ture. Only rise in titer signifi-sores, encephalitis complement fixation. cant. Most adults have antibodies.

Herpangina, pleurodynia, Coxsackie A Throat washings, stools. t Spinal fluid. Virus isolation, Many different virus types. Tis-aseptic meningitis and B Blood serum acu~e and convalescent. * neutralization, C-F. sue culture. Suckling mice.

Inclusion blennorrhea Large virus Epithelial scrapings of conjunctiva of Stained smear, egg Elementary bodies seen.

lower lid. inoculation. Occurs especially in newborn.

Infectious Unidentified Blood for white blood cell count. WBC and differential, Atypicallymphocytes.

mononucleosis Blood serum, acute and convalescent. * Paul-Bunnel test. Rising heterophil agglutinationtiter.

Influenza Influenza viruses Throat washings. t Virus isolation, ]n eggs, mice, or tissue culture.A, B, C (several Blood serum, acute and convalescent. * hernagglutination- Only greater than four-fold in-sublypes) inhibition, C-F. crease in titer diagnostic.

Ker atoconjW1ctivi tis, Adenovirus, Swabs or scrapings from conjunctiva. Virus isolation, Tissue culture.

epidemic type B Blood serum, acute and convalescent. :\< virus neutralization.

Lymphocytic LCM virus Blood serum, acute and convalescent. :\< Virus neutralization. Only rise in titer diagnostic.

choriomenin~tis

Lymphopathia LGV virus Pus o:r:tissue biopsy. t Skin test (Frei), Elementary bodies.venereum Blood serum, acute and convalescent. * virus isolation. In eggs and mice.

Mumps Mumps virus Saliva, spinal fluid. t Virus isolation, In eggs.Blood serum, acute and convalescent. * hemagglutination- Only rise in titer significant.

inhibition, C-F.

Meningitis. aseptic Coxsackie, ECHO, Spinal fluid. blood serum, acute and Virus isolation, Tissue culture. eggs. suckling

(viral)H.

simplex, polio, convalescent. * neutralization, C-F. mice.others

=m::.a PAP; aee::erespiratory disease

lARD)Psittacosis

_ Co_=: n.e:::.a.gg~.

:ac: :.'";e a = : . c . ~escen:...* ~ isolation. C-F.

virus neutralization.

Sputum, blood. t Virus isolation,Blood serwu, acute and convalescent. * com lement fixation.Brain tissue (autopsy). Smears for Negri

bodies.

Virus isolation.

Whole blood. Rickettsia isolation,

Blood serum. acute and convalescent. * complement fixation,Weil-Felix test.

Spinal, ventricular fluid, lymph node. Toxoplasma isolation,

In mice.

In guinea pigs.

Only 'rise in titer significant.

For results see chart p. 295.

In mice.


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may be specifically inhibited by antiserum. This reaction

forms the basis of a number of diagnostic tests for viral in-

fections.

To standardized amounts of virus, serial dilutions of the

serum under test are added (after heating the serum for 30

minutes at 56C.), and the mixture is shaken well. An equal

amount of 0.5% suspension ofhuman Type 0or chicken eryth-

rocytes is added and the mixture incubated at room temper-

ature for 45 to 60 minutes. Care must be taken not to disturbthe test tubes or racks. The tubes are then read for agglu-

tination of the red blood cells. The titer of a given serum is

defined as the highest dilution of serum which effects com-

plete inhibition of red cell agglutination.

Commonly Used Serologic Tests for Syphilis (S. T. S. ):

The tests below are accepted in the 1955 Manual of the

Public Health Service, U. S. Department of Health, Education,

and Welfare (Serologic Tests for SyphiliS, Public Health

Service Pub!. No. 411, U. S. Government Printing Office,

Washington 1955). Others are occasionally used. Adherence

to the published standard method is essential in obtaining re-

liable results.

Ifmore than one test is desired it is customary to per-

form one complement-fixation and one flocculation test. The

greater the agreement between different tests and the results

ofdifferent laboratories, the more confidence may be placed

in reports ofthese serologic findings. The different technics

estimate the presence of reagin, not of specific antibody

directed against T. pallidum.

A. Complement-Fixation Test: Kolmer-Wasserman.

B. Flocculation Tests: Hinton, Kahn, Kline, Mazzini,

APHA Reference Test.

C. VDRL (Ven.Dis.Ref.Lab.).

See the above manual for the standard technic ofthese

tests.

Antigens for S. T.S.:

The causative agent of syphilis, T. pallidum, has not yet

been grown in vitro. Thus antigens prepared from virulent T.

pallidum are not available. Experimentally, antigens from

the Reiter strain oftreponemes appear to have significant

specificity in complement -fixation test s, but they are not yet

widely used.It has been found empirically that sera from syphilitic in-

dividuals will flocculate or fix complement in the presence of

alcoholic extracts ofbeef heart muscle to which cholesterol

and lecithin have been added. Such standardized extracts

~rdiolipin-Iecithin antigens) are employed in most S. T. S.

...sedfor the assay of reagin. Living treponemes from animal

:es'ons are employed in the TPI (see p. 300).

?:'J.ids to Be Tested by S. T. S.:

A. Patient's Serum: Draw 5 to 10 m!. of sterile venous blood

and permit to clot in dry sterile test tube. Blood samples

should not be taken during high fever or soon after an-

esthesia or alcohol intoxication, for these and other con-ditions (see below) tend to give false-positive reactions.

Centrifuge the clotted blood and remove the cell-free

serum. Store in refrigerator until test can be performed.

Just prior to test the serum is inactivated at 56C.

(133F.) for 30 minutes to destroy complement.

3. pinal Fluid: Collect 3 m!. spinal fluid in sterile test

tube, free from blood cells (see p. 247).

:::Zerpretation of Results ofS. T. S.:

_. In untreated adults the S. T. S. will become positive in 60

to 80%of cases in the primary stage (four to eight weeks

after exposure), whether primary lesions are present ornot. Ifmanifest secondary lesions develop, the blood

:est will be positive in 90 to 100%ofthe cases and the

spinal fluid in 30 to 70%. In late (tertiary) lues, the blood

~s positive - if visceral lesions are developing - in 60 to

80%of cases. If there are no visceral lesions but C. N. S.

involvement, the spinal fluid is a better guide. Note,

:"owever, that in tabes dorsalis, both blood and C.S.F.

!:lay be negative in the presence of progressive symptoms

and signs. In general paresis both blood and C. S. F. are

almost always positive.

_. :n the newborn, and in infants up to ten weeks, the blood

:est may reflect the mother's reaction and does not

:1ecessarily indicate infection of the infant. It is

:,nportant, therefore, to follow titers of infants born of

mothers with positive serology but without evidence of

activity ofthe disease (e. g., adequately treated).

:. Antiluetic therapy usually results in gradual reversion

0: positive serologic tests to negativity. This, however,::lay take many months. A positive blood test in an ade-

".lately treated individual is therefore no justification for

ex:ending or repeating therapy unless there is evidence of

activity ofthe disease or rise in complement fixation

:::er. Antiluetic therapy, particularly with penicillin,

administered to darkfield-positive, seronegative individ-

:zals often interferes with the development of a positivecomplement fixation test.

= . Anticomplementary Specimens: Occasionally blood sam-:l2.esare encountered which fix complement in the absence

.. beef heart antigen. Such specimens cannot satisfactor-


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ily be examined by the complement fixation test. This

occurs particularly in hemolysed or contaminated speci-

mens. Such samples can be submitted to precipitation or

flocculation tests. "Anticomplementary" meanS neither

positive nor negative result.

E. Biologic false-positive serologic reactions for syphilis

occur in 0.01 to 0.1% of all positive tests. Commonly

they are weakly positive (except in yaws, pinta, and

bejel, where they are the same as in syphilis). .

Such biologic false-positive reactions may occur In

yaws, pinta (mal del pinto), bejel (all closely related

spirochetal infections), 50 to 100% of cases; leprosy, 40

to 70% of cases; infectious mononucleosis, 10 to 50% of

cases; malaria, 10 to 40% of cases; rat-bite fever and

relapsing fever, 20 to 50% of cases; all protozoan and

viral infectious diseases, occasionally; all diseases

associated with abnormally high globulin levels; all im-

munization and vaccination procedures; any fever of long

duration; all" collagen diseases." .

Unless supported by clinical findings, never accept the

result ofa single serologic test for syphilis as proving thediagnosis. Always request at least two examinations. Con-

sider conditions commonly responsible for biologic false-

positive reactions.

Quantitative S. T. S.:

Any serologic test for syphiliS may be carried out with

dilutions of serum, and the highest dilution giving a +++ + re-action may be reported. This permits evaluation of trends in

serologic response (result of therapy, indication of relapse,

follow-up of infants).

Number ofunits = serum dilution giving ++ ++ reactionx

4.

Treponema Pallidum Immobilization Test (TPI):

Serum from individuals with syphilis contains an antibody

which results in the rapid immobilization of actively motile

T. pallidum. This test has the greatest diagnostic specificity

because it measures a true antibody against the etiologic

organism of syphilis rather than the development of reagin

against a lipid extract of mammalian tissue which shows only

accidental relationship to the presence of syphilis. There-

fore, TPI can conclusively differentiate between biologic

false-positive tests and true syphilitic positives. Unfortu-

nately the test is not easy to perform and is available only in

a few central laboratories.

Technic: To dilutions of the test serum add a suspension

of actively motile T. pallidum freshly extracted from the

testicular chancre of an infected rabl:>it. A similar mixture

containing normal human serum is used as a control. Observe

:.::ese preparations microscopic

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