studies · studies ofhemolytic streptococcal infection ii. theserological reactions of the blood...

STUDIES OF HEMOLYTICSTREPTOCOCCALINFECTION

II. THE SEROLOGICALREACTIONSOF THE BLOODDURINGERYSIPELAS1

By WESLEYW. SPINK AND CHESTERS. KEEFER(From the Thorndike Memorial Laboratory, Second and Fourth Medical Services (Harvard),

Boston City Hospital and the Department of Medicine, Harvard Medical School, Boston)

(Received for publication September 5, 1935)

As a part of an investigation of the variousserological reactions that occur during and afterhemolytic streptococcal infections, we have studied30 patients with erysipelas. In a previous paper(1), various important factors in the epidemiol-ogy, prognosis and course of the disease in pa-tients with erysipelas were discussed. At thistime, we present information obtained from astudy of the antistreptolysin (antihemolysin),antifibrinolysin and the streptococcidal power ofwhole defibrinated blood. In addition, the com-plement of the blood serum was titrated, agglu-tination reactions against the organism derivedfrom the patient, and skin reactions to "Dick"toxin and streptococcal nucleoprotein were in-vestigated.

METHODSOF STUDY

All of the patients were studied while theywere under observation in the Boston City Hos-pital. After their illness had subsided and afterdischarge from the hospital they were followedfor periods of one to eight months. B. hemolyticstreptococci were isolated from all of the patients.When it was not possible to obtain the organismsdirectly from the lesion, they could be obtainedfrom the nasal secretions. It was of interestfrom the standpoint of the mode of invasion andpathogenesis of the infection that many of thepatients with erysipelas unassociated with a woundhad had a preceding acute follicular tonsillitis oran acute rhinitis from which B. hemolytic strep-tococci could be isolated with regularity. This isin agreement with the studies of Holmes (2).

Antistreptolysin. The antistreptolysin titer ofthe blood serum of the patients was determinedby the method of Todd (3) and examinationswere carried out in the same manner as that re-

'This work was aided, in part, by a grant from theMilton Fund and Clark Bequest of Harvard University.

corded in our previous studies (4) of patientswith rheumatic fever and rheumatoid arthritis.The results are expressed in units, a unit beingthe amount necessary to inactivate 0.5 cc. of astreptolysin solution of such potency that 0.2 cc.would completely hemolyze 0.5 cc. of a 5 per centsuspension of washed sheep cells in normal salinesolution. A serum was recorded as containing100 units per cc. when 1 cc. of the 1: 100 dilutionwas the least amount necessary to inactivate 0.5cc. of standard streptolysin solution.

Antifibrinolysin. The method of Tillett andGarner (5) was used in performing this test.The organism that we used for the production offibrinolysin was the same (CO) as that we haveemployed previously (6). The results were re-corded in degrees of resistance according to theclassification suggested by Tillett, Edwards andGarner (7). Control samples of blood plasmawere tested every day, and complete dissolutionof the plasma clot in all of our control patientstook place in less than one hour.

Streptococcidal power. The streptococcidal ac-tion was studied in the whole defibrinated bloodby the method of Todd, as employed by Ward(8) and Finland and Sutliff (9) in studying thepneumococcus. Different dilutions of an 18-hourbroth culture of the hemolytic streptococcus wereadded to 0.5 cc. of defibrinated blood in a smallpyrex tube. The tubes were sealed in a gas-oxygen flame and placed in a machine which ro-tated the tubes end to end within the incubatorfor 24 hours. At the end of that time the tubeswere opened, and cultures were made to determinewhich tubes continued to contain live organisms.The number of organisms in the various dilutionswas determined by plating 1 cc. of the dilutionsof 10a, 10T and 108. As a rule, in the 10-7dilution there were from eight to 24 organisms.

As controls, the patient's own organism was21

WESLEYW. SPINK AND CHESTERS. RtEEFER

mixed with the whole defibrinated blood of anindividual without streptococcus infection; theblood from the same individual was used eachtime the test was done.

In addition to studying the killing power ofwhole blood by means of the patient's own or-ganism, another virulent strain of hemolytic strep-tococcus, obtained from a case of meningitis, wasused repeatedly in each case. The purpose of thiswas to determine whether the killing power ofblood of patients with erysipelas was the sameor different for their own strains of streptococciand a strain of streptococcus isolated from anindividual suffering from a streptococcal infectionother than erysipelas.

The same procedure was carried out with thecontrols. The results are recorded in accordancewith the maximum number of organisms killedby 0.5 cc. of blood. That is to say, if there wasgrowth in the tube containing 0.1 cc. of a 10-dilution and none in the tube containing 0.1 cc. ofthe 10-2 dilution, it was recorded-killing powerin 10-2.

Skin tests. The ability of the patient's skin toreact to one skin test dose of Dick toxin and to0.01 mgm. of nucleoprotein derived from a strainof hemolytic streptococcus (NY5) was tested. Areaction, 1 cm. in diameter, was considered posi-tive.

Complement. The titer of the complement ofthe patient's blood serum was determined at dif-ferent times during the course of the disease.This was done by adding different amounts of thepatient's blood serum to 0.5 cc. of sensitized sheepcells and placing the mixture in the water bathat 370 C. for one hour. The smallest amount ofserum which was required to effect complete he-molysis of 0.5 cc. of sensitized sheep cells wastaken as the amount of complement present.

Sensitized sheep cells were prepared by mixingequal quantities of diluted amboceptor and a 5per cent suspension of washed sheep cells andplacing the mixture in the water bath at 370 C. forthirty minutes.

The stock amboceptor that was used in thesetests was diluted in accordance with its potencywhich had been determined by means of previoustitrations against sheep cells and complement. Inall of the tests reported, 0.1 cc. of the stock ambo-

ceptor was diluted to 25 cc. with physiologicalsalt solution, and used on the same day.

Agglutination tests. Agglutination tests weredone with the organism obtained from the patientand with the NY5 strain of a hemolytic strepto-coccus. All tests were carried out with organismsthat had been killed at 560 C. in accordance withthe technique previously described (10).

RESULTS

Antistreptolysin. Isolated observations showedthat the antistreptolysin titer of the blood serumof patients with erysipelas varied from 80 to10,000 units. This is in accord with our previousobservations (4) in patients with erysipelas. Ofgreater importance than isolated observations werethe changes in the titer that occurred during thecourse and following the recovery from the dis-ease. In no case did we fail to observe an in-crease in the antistreptolysin titer of the bloodserum as the disease progressed. The magnitudeof the increase is illustrated in Figure 1. In somethe amount of increase was slight, in others it wasconsiderable. When the serum of individual pa-tients was tested repeatedly over a considerableperiod of time, it was found that the titer of theantistreptolysin not only increased during thecourse of the disease but that it frequently re-mained elevated above the original determinationfor some weeks or months after the acute infec-tion subsided. The highest titer was usuallyreached within the first 20 days after the onsetof the illness, and, in most cases, it was obtainedby the 10th to the 15th day. In no case did weobserve an increase in the titer after the 20th day.Once the peak had been reached, increases in thetiter were not observed unless there was a rein-fection such as a recurrence of the erysipelas.The titer of the serum remained above the levelof the first observation for periods varying from40 days to six months. The titer of the antistrep-tolysin content of the serum at various times dur-ing and after the infection is charted in Figure 2.In all of the cases in which the titer remainedabove 1,000 units after the 20th day of the dis-ease, there were suppurative complications due tothe hemolytic streptococcus. Moreover, the pa-tients who had 500 or more units before the 10thday invariably gave a history of hemolytic strep-

22

HEMOLYTIC STREPTOCOCCALINFECTION. II

10000

8000

6000

-

o 4000I..

w

,, 2000

< 1000S&

a-

88

60

400

200 a aZ 'tt

FIG. 1. THE ANTISTIREPTOLYSIN IN PATIENTS WITH ERYSIPELASEach pair of dots represents the lowest and highest antistreptolysin

content of the blood observed during the course of the disease.

10000

8000

0

0 0

* *0 a

* .e

0 000 0 00

40 0 0 *p

*# of -

*O. 0 0*# 0

00,0:*0

0

.

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0 0

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0

10 20 30 40 50

DAY OF DISEASE

FIG. 2. THE ANTISTREPTOLYSIN TimR AND THE DAY OF THE DISEASEEach dot represents the antistreptolysin content of a specimen of blood serum.

23

, 6000

3-Io 4000I.-

w

I- 2000

to14 1000

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z

600

400

200

0 0 0*

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WESLEYW. SPINK AND CHESTERS. KEEFER

tococcus infection before the onset of the erysipe-las.

Antifibrinolysin. Of the 30 patients studied inthis series of cases, 24 developed maximum re-sistance to fibrinolysin. The results obtainedwere in general agreement with our previous ob-servations of this test in erysipelas, and are inaccord with the results recorded by Tillett (11).Our results in 55 cases are recorded in Table I.

Results of resistanceTABLE I

to fibrinolysin oftococcus

hemolytic strep-

Mumm HiEgh Diou Dmu NOresisMnce rOWIsisttnoe in tio i redistance.

Number No diuolo. Diuolutlon 3 to 8 1 to 3 Dieolutionof oase tlonlI-n in 8to 24 hous hm inIthoar

24 hours hours us ho 3 .1hour______++++ +++ ++ + _

Presentsl 30 24 1 3 2 0

Preioussres 25 22 0 2 1

65 46 1 5 3

In the present group of 30 patients, 15 showedmaximum resistance to fibrinolysis of their coagu-

lated plasma when they were first seen. Ninepatients, who showed no resistance when theywere first admitted, developed it during observa-tion, whereas the remaining six individuals never

developed maximum resistance. Once it ap-

peared, it persisted for periods of time varyingfrom 8 to 150 days. In the patients who didnot show resistance to fibrinolysis when they were

first seen, the day of the disease varied from thesecond to the ninth day, and resistance appearedbetween the fifth and 14th day. If no demon-strable resistance was present at the onset of theerysipelas, we failed to observe the developmentof complete resistance before the fifth day of thedisease.

The 15 patients showing maximum resistancewhen they were first examined require comment.These individuals were seen from the second tothe eighth day of their illness. In five of them,there would have been ample time for the devel-opment of resistance, as judged from the studyof individuals who were seen early in the course

of the illness. In the other 10 cases, whichshowed maximum resistance before the fifth dayof the attack of erysipelas, the presence of an

active hemolytic streptococcal infection of thethroat and nasopharynx prior to the onset oferysipelas was the most likely explanation forthe presence of antifibrinolysin. This conclusionreceived support from their clinical history. Itwas of further interest that erysipelas could occurin spite of the presence of maximum resistanceto fibrinolysis. This fact is emphasized by theobservation of the presence of resistance at theonset of recurrences of erysipelas. Wealso haveobserved cases in which resistance to fibrinolysinhad disappeared and then reappeared following arecurrence of a streptococcal infection. It seemsevident, then, that resistance to fibrinolysin is aresponse to a hemolytic streptococcal infection,and once it becomes established it may persistfor periods of time varying from eight to 150 days.

STREPTOCOCCIDALPOWEROF THE WHOLE

DEFIBRINATED BLOOD

1. Controls: Patient's organism and whole de-fibrinated blood of controls. One-half a cubiccentimeter of a normal individual's whole de-fibrinated blood was mixed with different dilutionsof a 24-hour broth culture of the hemolytic strep-tococcus isolated from the patient and incubatedas described under methods of study. The sameindividual was used as a control every time thepatient's blood was studied, and in this way theresults could be compared. It was found thatthe streptococcidal power of the blood of the con-trols did not vary more than a dilution found intwo tubes, i.e. 100-fold dilution when the numberof organisms in the dilutions was controlled. Forthis reason, we believed that variations in thestreptococcidal power of the patient's blood wouldhave to be greater than the dilution of two tubesin order to be of significance. In other words,an increase greater than 100 times the killingpower of the first observation was necessary be-fore we considered that the patient showed asignificant increase in his streptococcidal power.In case there was no. evidence of an increase ofstreptococcidal power as defined above, we con-sidered that the patient had a higher streptococ-cidal power than the control if his blood wereable to kill more than 100 times as many organ-isms at any time during the period of observations.

When the standards described above were used,

24


it was found that there was considerable variationin the killing power of the blood of the controlswhen the different organisms were used. Themaximum number of organisms killed by the bloodof the controls during the period of observationis shown in Column B of Figure 3.

id1

1020

go- 'a: 10

U.40

z lZ 10540

a lot

1O7

NO KILLINGPOWER

of the increase in streptococcidal power duringthe course of the disease and the maximum num-ber of organisms killed. When the results werestudied to determine the difference in the strep-tococcidal power of the blood in the patients andin the controls, it was found that eight patients

FIG. 3. THE MAXIMUMSTREPTOCOCCIDALPOWE IN PATIENTS AND CONTROLSEach dot in Column A represents the maximum streptococcidal power of a

specimen of whole blood against the patient's own organism. Each dot in Column Brepresents the maximum streptococcidal power of whole blood in controls against thepatient's organism. Each dot in Column C represents the maximum streptococcidalpower of a specimen of whole blood from patients with erysipelas against anotherstrain of hemolytic streptococcus derived from a non-erysipelas infection. Each dotin Column D represents the maximum streptococcidal power of a specimen of wholeblood from control individuals using the same strain as in Column C.

2. Patient's own organism against patient'swhole defibrinated blood. When the streptococ-cidal power of the patients' blood was studied,it was found that they were able to kill varyingnumbers of their own organisms. Column A ofFigure 3 shows the maximum number of organ-

isms killed by the patient's own blood and ColumnB the maximum number killed by the controls.In Figure 4, the change in the streptococcidalpower on repeated examinations is charted. Gen-erally speaking, the patients showed greater strep-tococcidal power than the controls; this was true

showed more than a 100-fold increase in theirstreptococcidal power, 13 a 10-fold to a 100-foldincrease, and eight no change. Upon the remain-ing patient only one observation was made. Theincreases in streptococcidal power were observedfrom the 8th to the 81st day of the diseaseand did not coincide with the recovery of thepatient. From these observations it was plainthat during the course of the disease it could beshown that the streptococcidal power of the wholeblood of some individuals increased considerably,while in others the changes were less striking.

A B C D

00*0*0000 *00 *.0.009*.*. *0e00*.0 *00

*00000*0*0 **000 .**000 *000*0000

*0S@ 0000. 0000.0 ***.00*000.000000

* 00*0000000 000 *000

000.000

* 0

25


Despite the fact that a number of patientsfailed to show very significant increases in strep-tococcidal power during the period of observation,

101I o-2102

CA

la.

Ot 1o-3C

:0

zJ 1651d7

n.--.a

NO KILLINGPOWER

44I

I11l1110

likely due to the presence of an active hemolyticstreptococcal infection.

Whole defibrinated blood of patients and con-

"' ,

Io 9

8 8 ooCOo

FIG. 4. THE MAXIMUMAND MINIMUM STREPTOCOCCIDALPOWEROF THE WHOLEBLOOD INPATIENTS AND CONTROLS

Each pair of solid dots represent the maximum and minimum streptococcidal power of theblood in patients with erysipelas using the organism derived from the patient. Each pair ofcircles represent the same determination in controls.

it was noted repeatedly that the patients had ahigher streptococcidal power than the controls.Thirteen of them were able at all times to killfrom 1,000 to 10,000 times as many organismsas the controls, 10 killed 100 times as many orless, five the same number as the control and, inone, the control was better than the patient.That is to say, in some patients there was anactual increase in the whole blood killing powerof the patients' blood during the course of thedisease; in other cases, there was evidence thatthe streptococcidal power of the patients' bloodwas greater than the controls although no strikingincrease was noted during the period of obser-vation.

These observations indicate that the killingpower of whole blood of patients with erysipelasis frequently increased above that of controls, andthat the increase in the killing power is most

trols and an organism derived from streptococcalmeningitis. In addition to studying the strepto-coccidal power of the blood with organisms de-rived from the patients, another strain of hemo-lytic streptococcus was obtained from a case ofstreptococcal meningitis and studied in connectionwith the blood of patients with erysipelas andcontrols. The maximum killing power of theblood serum in each case is recorded in Figure 3.Column C represents the results in patients witherysipelas and Column D, the results in the con-trols. By and large, the patients with erysipelasshowed somewhat greater streptococcidal powerfor this organism than the controls. It is to benoted, however, that the different controls pos-sessed some streptococcidal power against thisparticular strain of hemolytic streptococcus, indi-cating that the ability to kill certain strains of

26

, 044

HEMOLYTIC STREPTOCOCCALINFECTION. I2

hemolytic streptococci is possessed by some nor-mal individuals without infection.

Results of complement titration. Since it isgenerally believed that complement contributes tothe intracellular destruction of organisms by ren-dering them susceptible to phagocytosis, it wastitrated in the blood serum of the patients studiedby us. The purpose of this was to determinewhether there was any correlation between thekilling power of whole blood and the amount ofcomplement present in it and, secondly, whetherthere were any consistent changes during the

sipelas to render the organisms susceptible tophagocytosis.

The fluctuations of complement during thecourse of the disease proved of interest. It hasbeen stated that the complement of normal non-infected individuals fluctuates only slightly be-tween 0.03 and 0.06 cc. This is not in agree-ment with our observations. Wehave found widevariations in different individuals when the titra-tions are done repeatedly. Figure 6 illustrates thevariation of the titer in four individuals whoseblood was titrated repeatedly over a period of from

@*0 *-a0 0s

cc2I* *0 * *. * * * * 0000@*a~~ *.f *..** * -* . ** ** ** *

104 v0

z 10 0 0 000 00

0j-6ao

167 ' 0 0 0 0* 0

NO KILLING *9* *POWER

.01 .02 D3 .04 .05 .06 .07 .08 .09 .1 .2 .3 .4 +.4C.C.

FIG. 5. THE STREPTOCOCCIDALPOWEROF THE WHOLEBLOODAND THE COMPLEMENTTITER oFTHE BLOODSERUM

Each dot represents the streptococcidal power of a specimen of whole blood and the com-plement titer of the blood serum from the same individual.

course of the disease. The results have beencharted in Figure 5.

It was not possible to demonstrate any definitecorrelation between the amount of complementand the streptococcidal power of the whole de-fibrinated blood. From these observations, itwould appear that in most cases at least there wassufficient complement present in cases of ery-

one to two years. While these individuals didnot show fluctuations of more than 0.1 cc. on re-peated examinations, it is obvious that normalindividuals may show great differences in theamount of complement present. This must betaken into account in any interpretation of com-plement titers.

During the course of erysipelas, the titer of the

I

27


complement in patients with erysipelas decreasedmore than 0.1 cc. in nine, increased more than 0.1cc. in five and remained the same in 16. Fromthese observations it can be concluded that during

0.01

0.1

0.2 ob-oo--6 o-o--o-d

o 0.3

0.4

+ 0.4

in dilutions varying from 1 to 40 to 1 to 80, whilesix agglutinated NY5. In two patients showingthe highest titers there were suppurative compli-cations. From these observations, it is possible

FIG. 6. THE VARIATIONS IN COMPLEMENTTITER IN NORMALINDMDUALS

Each line indicates the fluctuation of the complement titer in a normal individual followed over

a period varying from one to two years.

the course of erysipelas the complement of some

patients may fluctuate in greater amounts thanis seen in controls without infection. In our ex-

perience, however, sharp increases or decreasesas indicated by the titration could not be corre-

lated with an improvement or an exacerbation ofthe infection. The magnitude of the fluctuationsobserved in infected individuals is shown inFigure 7.

Agglutination tests. The blood serum frompatients with erysipelas was tested for agglutininsagainst their own organisms at various intervalsduring and after the course of the disease. Inaddition to using the patient's own organism, theNY5 strain of hemolytic streptococcus was usedas an agglutinogen. Only three of 30 patientsshowed agglutinins against their own organisms

to state that in our experience at least, agglutininsappear in the serum irregularly and the titer isnot high. This is precisely what we (10) foundin previous observations.

Skin tests. Results of Dick test. Only one ofthe 30 patients showed a positive skin reactionfollowing the injection of one skin test dose of" Dick " toxin into the skin. The others yieldednegative results. When the nucleoprotein de-rived from the Streptococcus scarlatinae was used,it was found that 24 reacted in a positive manner

and six were negative. Of the 24 who reacted,20 of them showed a positive reaction to 0.01cc. when first tested, while four more showedpositive reactions to a second injection of thesame material given five to seven days after thefirst.

*

a./ \'

o

&-o.,d 6

II

28


DISCUSSION

From the data presented there is considerableevidence that during and following erysipelasthere is an increase in the antistreptolysin andantifibrinolysin of the blood. In addition, in aboutone-third of the cases, the streptococcidal powerof the whole blood increases. While there is noprecise relation between the time of recovery andthe demonstration of increasing amounts of theseantibodies in the blood, there seems to be littledoubt that their presence indicates a response onthe part of the host to the streptococcal infec-tion.

0.01

0.1

0.2Li

0.3

0.4

+0.4

unless rendered susceptible by the presence ofantibacterial substances. Moreover, it can be dem-onstrated that these different special propertiescall forth specific immunological responses whichare independent of one another. It was for thesereasons we studied the different kinds of responsesimultaneously.

In view of the multiple serological reactionsthat can be demonstrated in patients with hemo-lytic streptococcal infection, it is naturally difficultto decide whether one response is more importantthan another in bringing about recovery. Recov-ery probably results from a summation of a variety

FIG. 7. THE MAXIMUMAND MINIMUM AMOUNTSOF COMPLEMENTEach pair of dots represents the maximum and minimum amounts of comple-

ment observed during the course of the disease.

The complement was present in varyingamounts and, in most cases, seemed to be adequatefor the phagocytosis of streptococci as indicatedby the streptococcidal test.

From a consideration of the results of numer-ous investigations, it is generally believed thathemolytic streptococci are capable of elaboratingtoxins, hemolysins, leukocidins and fibrinolyticsubstances. In addition, they resist phagocytosis

of processes which are capable of keeping theinfectious process localized and destroying or lim-iting the growth of the organisms in the tissues.Of all these reactions, the presence of antibodiesthat aid in the phagocytosis and destruction ofthe organism would seem to be of the greatestimportance.

Recent investigations by Hare (12) of hemo-lytic streptococcal infections, especially those oc-

:1 1 ri I :11

I 4 4

29

6

: 04 4

41:4


curring in the puerperium, are significant. Sincethese infections are commonly localized in onesmall area, that is, the uterus and its immediateneighborhood, the results he obtained from thestudy of the bactericidal power of the blood dur-ing this type of infection were of interest in thelight of the present investigation of erysipelas.In Hare's experience, the patients with hemolyticstreptococcal infections in which the process waslocalized to the uterus or its immediate neigh-borhood, showed a normal bactericidal power ofthe blood early in the course of the disease. Therewas a tendency in some cases for this to increase,but it was not a constant finding so that he be-

DAYS5 10 I5 20 44

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REACIIONS DURING AND FoLLov

lieves that it was doubtful whether the absenceof more widespread invasion was due to a highdegree of general immunity. In the patients withgeneralized infection who recovered, the bacteri-cidal power of the blood was much greater thannormal, and in patients with generalized infectionwho died, the bactericidal power was greater thannormal in about one half the cases. Hare's re-

sults are in general agreement with our observa-tions in patients with erysipelas and other strep-tococcal infections. That is to say, in some pa-tients with a localized infection, recovery mayoccur without any demonstrable increase in thebactericidal power of the blood. In other patients,an increase in general resistance was shown, andthis occurred even in the presence of invasion ofthe blood stream.

Two types of response that may be observed inpatients with erysipelas are illustrated by Figures8 and 9. The various observations made in Case2 are charted in Figure 8. During the courseand following the illness in this case there was

OF OSURVATIONO 60 60 100- 120 140

IANGES IN THE VAIOUS SEROLOGICALWING AN ATrACK OF ERYSIPELAS

an increase in the titer of antistreptolysin, anti-fibrinolysin and the killing power of whole blood.Agglutinins appeared in several patients, espe-cially in the presence of suppurative complications.It was striking that the increased streptococcidalpower and the antistreptolysin persisted for aperiod of six months, whereas the antifibrinolysindiminished after 22 days.

30


Figure 9 illustrates the fact that erysipelas mayoccur in spite of the presence of increased re-sistance to fibrinolysis and good streptococcidalpower of the whole blood. It is obvious that theblood of the control possessed as great a strepto-

DAYS Or OBSERVATION5 10 15 20 25

104103

102

99

1000

X ,

DICK NIB.

£C"6. M"S. ME. MG

FIG. 9. CASE 3, SHOWINGTHE SEROLOGICALREACTIONS DURING THE COURSEOF THE

ILLNESS

eoccidal power for the organism causing the dis-ease as did the blood from the patient. This stateof affairs was observed upon five occasions andemphasizes the fact that some individuals possessstreptococcidal power of high degree againstcertain strains of hemolytic streptococci and stillremain susceptible to local infections produced bysuch organisms.

To explain the recovery of erysipelas, it hasbeen postulated that it is dependent upon: (1)the development of a local tissue immunity, and(2) a neutralization of toxin. These views re-quire comment.

Local immunity. It has been postulated, espe-cially by Gay and Rhodes (13), that there exists

a true local tissue immunity following local strep-tococcus infection or immunization. This state-ment was based upon clinical and experimentalobservations. They pointed out that it had beenshown in both man and in animals that the injec-tion of hemolytic streptococci into an area of skinwhich had been the site of erysipelas some weeksbefore may not be followed by an infection. Thisapplied not only to the previously involved areabut also to other areas of skin as well. Workingwith rabbits, Gay and Rhodes reported that fol-lowing recovery from erysipelas there was com-plete protection against intradermal reinoculation,irrespective of its locality, provided one waitedfor at least three weeks after the primary inocu-lation induced by previous intradermal infectionor immunization could be associated with a lackof protection against intravenous injection of or-ganisms. Conversely, intravenous immunizationprotected better against intravenous than againstintradermal injection. From these observations,they concluded that a true local tissue immunityexisted following the infection. It was inferredthat the fixed tissue cells of the skin themselveshad become immune to infection.

Rivers and Tillett (14) have demonstrated thatdermal tissues of a rabbit become more refractorythan normal skin to infection with hemolytic strep-tococci when they are infiltrated with normalserum, homologous serum or meat infusion broth,24 hours before inoculation. Of the three sub-stances, the most effective was the homologousimmune serum. The refractory state induced inthe tissues by the normal serum or the meat in-fusion broth was said to be non-specific in char-acter, whereas the amount of resistance producedby the immune serum that was greater than thatcaused by the normal serum was considered to bethe degree of local passive immunity conferredupon the tissues treated with the immune serum.It should be recalled that the tissues were moreresistant to the infection when the inflammatoryreaction had been induced before the inoculationof hemolytic streptococci. The precise mechanismby which this refractory state was brought intobeing remained obscure, but it was manifest thatlocal passive immunity could be induced withhomologous immune serum and existed longenough to prevent a local infection of the skin.

31


Amoss and Bliss (15) also reported that re-

peated injections of hemolytic streptococci in rab-bits were followed by a demonstrable humoralimmunity and, with the appearance of the anti-bodies, the skin became generally resistant toinfection with hemolytic streptococci. It was

found that the skin showed some resistance torepeated injection of microorganisms so that thereactions to second injections of the same micro-organisms were not so striking as the first infec-tions.

From the observations cited it is not absolutelyclear that local immunity of the tissues can existto the extent that bacteria are immobilized locallyand their growth suppressed without the existenceof humoral immunity. Indeed, from the experi-ments of Rich and McKee (16) with pneumo-

cocci, and others with different organisms, it wouldappear that fixation of organisms locally in theimmune animal is dependent largely upon thepresence of immune bodies in the blood serum

of the animal, rather than upon immunity of thefixed tissue cells.

Neutralization of toxin. The evidence that a

toxin is elaborated from the local lesion in ery-

sipelas is controversial insofar as its presence hasbeen detected in the circulating blood. Birkhaug(17) reports the finding of a soluble toxin in thecirculating blood of patients with erysipelas,whereas Francis (18) was unable to confirm thisobservation. There is ample evidence, however,that many strains of hemolytic streptococci whichare isolated from the lesions of erysipelas are

capable of producing toxin.Francis has reported, however, that early in the

course of the disease there were antibodies in thecirculating blood which were capable of neutraliz-ing the toxic filtrates of hemolytic streptococciisolated from the lesions when such filtrates were

tested on human skin. As the disease progressed,there was a tendency for patients to show in-creased reactivity of the skin to toxic filtrates ofthe organisms and a decrease in the ability of theserum to neutralize the toxic filtrate. The con-

clusions reported by Francis were to the effectthat recovery from erysipelas in adults was notdue to the neutralization of a circulating toxinthrough the development of an antitoxin, but itwas related to the development of allergy to prod-

ucts of the growth and dissolution of streptococciin the erysipelatous lesion.

From the present studies, we have no compar-

able or direct evidence bearing upon the abovequestion. Wehave found that 29 of the 30 pa-

tients gave no reaction to the Dick toxin and 26of the 30 patients reacted in a positive manner tothe nucleoprotein of a hemolytic streptococcusderived from scarlet fever and a strain whichhas a broad antigenic base (19). These reactionsgave no indication regarding the mechanisms ofrecovery, but indicate that many patients with ery-

sipelas are capable of neutralizing -Dick toxin andreact to the nucleoprotein of this organism (20)as do other individuals with hemolytic strepto-coccal infection.

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FIG. 10. CASE 10, SHOWINGTHE SEROLOGICALREACTIONSIN Two ATTACKS OF ERYSIPELAS

From our studies, there can be little questionthat during the course of erysipelas there is evi-dence of the appearance or increase of antibodiesagainst the different special properties of the he-

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32


molytic streptococcus. The intensity of the dif-ferent responses varied from case to case and, inmany, there was evidence of some humoral im-munity early in the course of the disease. It isnot inconsistent, then, to say that the presence ofsome resistance to infection as measured by strep-tococcidal power, may not protect against a locallesion provided the conditions for infection arefavorable; once the infection becomes establishedthere is frequently an increase in humoral im-munity which may aid in recovery.

Recurrent attacks. It is a matter of commonexperience to observe repeated attacks of erysipe-las in the same individual. It would appear thatone attack of erysipelas renders some individualsmore susceptible rather than more resistant tosubsequent infections, and, from our studies andthose of others (21, 22, 23, 24, 25), there can belittle doubt that this is the case.

lesion on both occasions; the clinical course wassimilar, and the second attack occurred in spite ofthe presence of the different antibodies above thenormal level.

The second type is illustrated in Figure 11.This patient has had, to our knowledge, eightattacks of erysipelas during the past two years,six attacks of facial erysipelas and two attacks oferysipelas of the legs. The attacks are abruptin onset, and within a few hours the local lesionoften, but not always, reaches its maximum in-tensity. Microorganisms cannot be isolated fromthe lesions themselves, but they are present con-stantly in the paranasal sinuses and the nasalsecretions. Moreover, attacks can be induced bythe injection of a toxic filtrate derived from theorganisms that are present in the nasal secretions.In view of the repeated attacks, the inability toobtain organisms from the lesions, and the pro-

DAYS OF osBSRVATION10 20 30 40 50 60 70 SO 100 120 140 160 1iS

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FIG. 11. CASE 1, SHOWINGTHE SEROLOGICALREACTIONSDURINGANDBETWEENREPEATEDATrACKS OF ERYSIPELAS

We have observed recurrent attacks of ery-sipelas of several types. The first is illustrated inFigure 10. In this case, the two attacks of ery-sipelas occurred within two weeks of each other,and hemolytic streptococci were isolated from the

duction of a typical attack by the injection of atoxic filtrate into a site some distance from thearea which becomes involved, there seems to belittle doubt that this type of reaction is due to aresponse of sensitized tissues to the products of

33


the hemolytic streptococcus. Figure 11 showsthat the repeated attacks of erysipelas occurredin spite of the fact that there was a certain degreeof resistance to streptococcal infection as evi-denced by the amounts of the different antibodies.

SUMMARYAND CONCLUSIONS

From a study of the clinical course of 30patients with erysipelas and the serological reac-tions that can be demonstrated during and fol-lowing the -disease, the following results wereobtained.

1. Erysipelas not infrequently follows a strep-tococcal infection of the throat and nasal passages.

2. The antistreptolysin (antihemolysin) of theblood serum increased during the course of thedisease, and frequently remained above the orig-inal titer for periods of 40 days to six months.The highest titer was usually reached within thefirst 20 days after the onset of the illness.

3. Twenty-four of the 30 patients developedmaximum resistance of fibrinolysin within the fiveto fourteen days of the illness. Once it appeared,it persisted for periods of time varying from8 to 150 days. While there was no precise cor-relation between the appearance of maximumresistance and recovery, we considered this re-action a response on the part of the host tostreptococcal infection.

4. The streptococcidal power of the bloodshowed that during the course of the diseasethere may be an increase in the titer of this prop-erty of the blood or it may remain stationary.Increases in killing power could not be correlatedwith recovery. It was considered to be a responseon the part of the host to the streptococcal in-fection.

5. The complement titer of the blood serumwas determined, and it was found that it in-creased more than 0.1 cc. in five, decreased morethan 0.1 cc. in nine, and remained the same in 16.Generally speaking, there was an adequate amountof complement present for phagocytosis.

6. Only one of the 30 patients reacted posi-tively to Dick toxin, whereas 24 patients showedpositive reactions to 0.1 mgm. of the nucleoproteinof hemolytic streptococcus.

7. Three patients developed agglutinins againsttheir own organisms in titers varying from 1 to

40 to 1 to 80. In two of these, there weresuppurative complications.

8. The mechanism of recovery from erysipelasis discussed in the light of the above findings.

Weacknowledge our thanks to Miss MarjorieJewell and Miss Eleanor Fleming for technicalassistance.

BIBLIOGRAPHY

1. Keefer, C. S., and Spink, W. W., Studies of hemo-lytic streptococcal infection. I. Factors influencingthe outcome of erysipelas. J. Clin. Invest., 1936,15, 17.

2. Holmes, C. R., Etiology of erysipelas; Its relation tothe nasal cavities and its destructive effects uponthe eye. Ann. Otol., Rhin. and Laryng., 1907, 16,457.

3. Todd, E. W., Antigenic streptococcal hemolysin. J.Exper. Med., 1932, 55, 267.

4. Myers, W. K., and Keefer, C. S., Antistreptolysincontent of the blood serum in rheumatic fever andrheumatoid arthritis. J. Clin. Invest., 1934, 13,155.

5. Tillett, W. S., and Garner, R. L., The fibrinol-tic ac-tivity of hemolytic streptococci. J. Exper. Med.,1933, 58, 485.

6. Myers, W. K., Keefer, C. S., and Holmes, W. F., Jr.,The resistance to fibrinolytic activity of the hemo-lytic streptococcus with special reference to pa-tients with rheumatic fever and rheumatoid(atrophic) arthritis. J. Clin. Invest., 1935, 14, 119.

7. Tillett, W. S., Edwards, L. B., and Garner, R. L.,Fibrinolytic activity of hemolytic streptococci.The development of resistance to fibrinolysis fol-lowing acute hemolytic streptococcus infections.J. Clin. Invest., 1934, 13, 47.

8. Ward, H. K., Observations on the phagocytosis of thepneumococcus by human whole blood. I. The nor-mal phagocytic titre, and the antiphagocytic effectof the specific soluble substance. J. Exper. Med.,1930, 51, 675.

9. Finland, M., and Sutliff, W. D., Immunity reactionsof human subjects to strains of pneumococci otherthan Types I, II and III. J. Exper. Med., 1933,57, 95.

10. Keefer, C. S., Myers, W. K., and Oppel, T. W.,Streptococcal agglutinins in patients with rheu-matoid (atrophic) arthritis and acute rheumaticfever. J. Clin. Invest., 1933, 12, 267.

11. Tillett, W. S., The occurrence of antifibrinolyticproperties in the blood of patients with acutehemolytic streptococcus infections. J. Clin. Invest.,1935, 14, 276.

12. Hare, R., Alterations in the bactericidal power of theblood which occur during hemolytic streptococcalinfections in the puerperium. J. Path. and Bact.,1935, 41, 61.

34


13. Gay, F. P., and Rhodes, B., Experimental erysipelas:Studies in streptococcus infection and immunity.IV. J. Infect. Dis., 1932, 31, 101.

14. Rivers, T. M., and Tillett, W. S., Local passive im-munity in the skin of rabbits to infection with (1)a filtrable virus, and (2) hemolytic streptococci.J. Exper. Med., 1925, 41, 185.

15. Amoss, H. L., and Bliss, E. A., Local immunity inexperimental erysipelas. J. Exper. Med., 1927, 45,411.

16. Rich, A. R., and McKee, C. M., A study of the char-acter and degree of protection afforded by the im-mune state independently of the leucocytes. Bull.Johns Hopkins Hosp., 1934, 54, 277.

17. Birkhaug, K. E., The etiology of erysipelas. Arch.Path., 1928, 6, 441.

18. Francis, T., Jr., Studies on pathogenesis and recovery

in erysipelas. J. Clin. Invest., 1928, 6, 221.19. Wadsworth, A. B., Serum therapy-Its value in

pneumonia, meningitis, scarlet fever and other

streptococcus infections. J. A. M. A., 1932, 99,204.

20. Myers, W. K., Keefer, C. S., and Oppel, T. W., Skinreactions to nucleoprotein of streptococcus scarla-tinae in patients with rheumatoid arthritis andrheumatic fever. J. Clin. Invest., 1933, 12, 279.

21. Amoss, H. L., Treatment of recurrent erysipelas.Ann. Int. Med., 1931, 5, 500.

22. Stevens, F. A., Chronic infectional edema. J. A. M.A., 1933, 100, 1754.

23. Dochez, A. R., and Stevens, F. A., Studies on thebiology of streptococcus. VII. Allergic reactionswith strains from erysipelas. J. Exper. Med.,1927, 46, 487.

24. Birkhaug, K., Erysipelas. VIII. Bacterial allergyto streptococcus erysipelatis in recurrent erysipelas.J. A. M. A., 1928, 90, 1997.

25. Amoss, H. L., Hansen-Pruss, 0. C., and Bliss, E. A.,Erysipeloid reactions as an allergic response. Tr.A. Am. Physicians, 1928, 43, 259.

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