INFECTION AND IMMUNITY, Dec. 1982, p. 1196-12020019-9567/82/121196-07$02.00/0

Vol. 38, No. 3

Streptococcal Neuraminidase and Acute GlomerulonephritisELIZABETH V. POTTER,2* MARTHA A. SHAUGHNESSY,2 THEO POON-KING,1 AND

DAVID P. EARLE2

Streptococcal Disease Unit, The General Hospital, San Fernando, Trinidad,1 and Section of Nephrology andHypertension, Northwestern University-McGaw Medical Center, Chicago, Illinois 606112

Received 1 June 1982/Accepted 16 September 1982

We examined the hypothesis that streptococcal neuraminidase may alter hostserum immunoglobulin G so that autoantibodies are formed which lead to immunecomplexes and acute glomerulonephritis. We confirmed the observation that T-type 4 and T-type 12 streptococci (both associated with acute glomerulonephritis)are the most likely of many types studied to produce neuraminidase. However, wedid not find this enzyme to be produced by any of 23 streptococcal strains isolatedfrom patients with nephritis, whereas it was produced by two strains from patientswith rheumatic fever and by one strain from a patient with scarlet fever. Also, wewere unable to find direct or indirect evidence of increased neuraminidase activityin the sera of six patients with acute glomerulonephritis when they were comparedwith the sera of six patients with acute rheumatic fever and with those of sixnormal subjects.

For some time, differences have been soughtamong the streptococci associated with acuteglomerulonephritis (AGN), acute rheumatic fe-ver, or pharyngitis without nonsuppurative se-quelae which might contribute to their respec-tive pathogenicities. None have been found inthe in vitro production of streptolysin 0, strep-tolysin S, hyaluronidase, DNAse B, M protein,or group A carbohydrate (10, 12). However,Davis et al. (1) reported that of several strepto-coccal T types from patients without AGN,mainly 4 and 12 produced neuraminidase invitro. Both of these types have been associatedwith AGN (2). McIntosh et al. (8) describedalteration in vitro of serum immunoglobulin G(IgG) by neuraminidase from streptococci orVibrio cholerae which made the IgG autoanti-genic in rabbits. Furthermore, these workerseluted antibody to neuraminidase-altered humanIgG from glomerular deposits of a patient whodied in the acute stage of AGN (17). Theyhypothesized that streptococcal neuraminidasehad altered host IgG so that autoantibodies wereinduced which formed complement-fixing im-mune complexes and lodged in the glomerularcapillaries.

This hypothesis accounts for the lag periodrequired for AGN to develop after streptococcalinfections and for the fact that streptococcalantigens have not often been demonstrated inthe glomerular lesions of streptococcal AGN. Ifthis hypothesis is valid, increased neuramini-dase production by streptococcal strains isolat-ed from AGN patients should be expected, aswell as direct or indirect evidence of neuramini-

dase in the sera of these patients. Direct evi-dence would be the ability of sera to free sialicacid from a glycoprotein substrate in vitro,whereas indirect evidence would include in-creased amounts of free sialic acid, decreasedamounts of bound sialic acid, and antibody toneuraminidase in the sera of these patients. Inthe present study, these factors were sought instreptococcal strains and in sera from patientswith AGN in Trinidad and were compared withthe same factors in streptococcal strains andsera from patients with rheumatic fever oruncomplicated sore throats.

MATERIALS AND METHODS

Streptococcal strains studied. Ninety-three strepto-coccal strains from patients with upper respiratoryinfections were obtained from Children's MemorialHospital, Chicago, Ill., during February and March of1980. Five strains from patients with nephritis or theirsiblings were obtained from the University of Chicago.All of these strains were kept on blood agar plates untilassayed for neuraminidase within 6 days of theirisolation. One M-type 12 strain (SF42) isolated byGriffith many years ago from a patient with scarletfever (14) had been subcultured, lyophilized, mouse-passed, and frozen many times before this study. Allof these strains were grouped, M-typed (9), and T-typed (18).

Fifty-five streptococcal strains were selected fromthose isolated 2 months to 15 years ago in Trinidadfrom patients with AGN, rheumatic fever, or sorethroats. These strains had been frozen soon afterisolation and were thawed and retyped for this study.Those strains that were stored for longer than 2 yearsor were no longer M-typable (or both) were blood-

11%

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from

STREPTOCOCCAL NEURAMINIDASE AND NEPHRITIS 1197

passed (an inoculum of 200 to 400 streptococcal unitsin 0.1 ml of Todd-Hewitt broth rotated in 0.4 ml offresh human nonimmune blood for 4 h at 37°C) fivetimes before testing for neuraminidase production.Serum samples studied. Serum samples were ob-

tained by centrifugation of fresh, clotted blood fromsix patients with AGN and six patients with rheumaticfever in Trinidad (15). Serum samples were also ob-tained from three well school children in Trinidad andfour healthy adults in Chicago. All sera were frozensoon after they were obtained and kept at -50 or-70°C until studied.Assays for sialic acid. The Warren assay (20) indi-

cates the presence of free sialic acid as thiobarbituricacid-reacting substances (TBARS) by spectrophoto-metric readings at 549 p.M. These substances will bereferred to as TBARS rather than as free sialic acid,since they may include lipid peroxides (5), deoxyri-bose (11, 20), and free DNA (3), which also producecolor in these tests. However, the peak optical densityof these other substances is 532 puM rather than 549,uM, the peak for sialic acid. Warren equation 2 (20),which was developed to correct for the presence ofother substances (deoxyribose sugars), was employedwhen readings were higher at 532 than at 549 p.M.Amounts of TBARS were read from a curve obtainedby plotting the readings at 549 p.M of five dilutions (1.9to 15.5 pug/ml) of purified N-acetylneuraminic acid(Sigma Chemical Co., St. Louis, Mo.) dissolved in 0.1N sulfuric acid. These lay in a straight line whenplotted on regular graph paper against their numericalvalues except for the most dilute preparation (1.9pg/ml). Since this was in the range of some of our testmaterials, Warren's equation 1 (20) for non-linearitywas applied to the readings. Positive controls for eachtest consisted of tubes with substrates (containingbound sialic acid) and neuraminidase from streptococ-cus 337 (a T-type 4 strain from Children's MemorialHospital), prepared as described below, or from Vibriocholerae (Behring Diagnostics, Summerville, N.J.), in0.05 M acetate buffer (pH 5.5) with 9 mg of NaCI perml and 1 mg of CaCl2 per ml. Substrates used for all ofthe tests were: bovine submaxillary mucin (BSM;Sigma Chemical Co.), 10 mg/ml in 0.05 M acetatebuffer (pH 5.5) with 0.02 M CaC12 and 0.15 M NaCl;and fetuin (Sigma Chemical Co.), 5 mg/ml in the samebuffer. Substrates also tested were chromatographical-ly purified IgG (Cappel Laboratories, Downingtown,Pa.) and a normal human serum. Negative controlsconsisted of each substrate in buffer, the readings ofwhich at 549 ipM were subtracted from the readings ofsubstrates plus test substances. The BSM, when inwater or acetate buffer, gave substantial readings at549 pFM (mean, 0.48) and 532 pM (mean, 0.40). WhenNaCl was added to the acetate buffer by the method ofDavis et al. (1), readings at both 549 and 532 p.M weremuch lower (0.04 and 0.05, respectively). Therefore,NaCl-acetate buffer was used for all of our assays.Fetuin in water, acetate buffer, or NaCl-acetate buffergave equal and very low readings at both wavelengths(50.02). Fetuin was also used in NaCl-acetate bufferfor all assays.The Jourdian assay (7) indicates the amount of total

and bound sialic acid from which free sialic acid maybe calculated. Amounts of total and bound sialic acidwere read from a plot of the readings at 630 p.M of fivestandards of purified N-acetylneuraminic acid (3.9 to

31.0 pRg/ml) dissolved in water. The points lay in astraight line when plotted on regular graph paperagainst their numerical values. Serum blanks (testserum plus periodate plus reagent without resorcinol)gave readings of only 0.005 at 630 FM in all cases.The Svennerholm assay (19) indicates the total

amount of sialic acid present. This was read from aplot of the readings at 580 ,uM of five to ten standardsof purified N-acetylneuraminic acid (15.5 to 155.0,ug/ml) dissolved in water. The points lay in a straightline when plotted on regular graph paper againstnumerical values of the standards until over 100 ,ug ofsialic acid were employed. Readings for the standards,for substrates, and for test sera in resorcinol werehigher at 580 than at 450 ,uM.

Assays for neuraminidase production by streptococci.Streptococci were grown for 16 h in Todd-Hewittbroth with 0.2% glucose and centrifuged. The superna-tants were dialyzed overnight against 0.1 M acetatebuffer (pH 5.5) with 0.01 M CaCI2, passed through a0.22 pum membrane filter (Millipore Corp., Bedford,Mass.), and stored at 4°C until used. These were testedfor neuraminidase activity by mixing 0.1 ml with 0.1 mlof one of the substrates, incubating overnight at 37°C,and testing for TBARS by the method of Warren (20).Negative controls consisted of each substrate in buff-er, the readings of which at 549 puM were subtractedfrom the readings of substrates plus streptococcalsupernatants. The glucose broth with buffer gavenegligible readings at 549 ,uM.

Assays for neuraminidase activity in serum samples.Direct evidence of neuraminidase activity in the serumsamples was sought by measuring by the method ofWarren (20) the TBARS released when 0.1-mlamounts of the serum samples were mixed with 0.1-mlamounts of glycoprotein substrates (BSM or fetuin)and incubated for 30 min. Any TBARS found by thesame technique in the serum samples plus bufferinstead of substrate or in substrate plus buffer wassubtracted.

Indirect evidence of neuraminidase activity in theserum samples was sought by measuring total andbound sialic acid by the method of Jourdian et al. (7)and calculating free sialic acid therefrom. A decreasedtotal or bound sialic acid or an increased free sialicacid would suggest neuraminidase activity. By usingthis resorcinal method of testing the serum mixturesrather than the thiobarbituric acid assay, we sought toavoid the chance of measuring lipid peroxides, deoxy-ribose, or free DNA as discussed above. Confusingreactions were more often apparent when serum sam-ples were tested than when streptococcal supernatantswere tested. Total amounts of sialic acid were alsomeasured in some sera by the method of Svennerholm(19).

Antibodies to streptococcal neuraminidase werelooked for in the serum samples by measuring theamount ofTBARS released from BSM or fetuin by thestreptococcal neuraminidase after incubation with andwithout the test sera. Fifty microliters of serum sam-ples or buffer was added to 50 p.I of streptococcalneuraminidase 337 and incubated at 4°C overnight;then, 100 pI of the BSM or fetuin preparation wasadded, and the mixture was incubated at 37°C for 30min, after which it was tested for TBARS released bythe neuraminidase in the presence and absence of thesera. Inhibition by the serum of release of TBARS

VOL. 38, 1982

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from

1198 POTTER ET AL.

from the substrates was considered suggestive evi-dence of antibody to neuraminidase.

RESULTS

Preliminary assays. When BSM, fetuin, chro-matographically purified IgG, and whole humanserum were assayed for sialic acid content, themethods of both Jourdian et al. (7) and Svenner-holm (19) indicated total amounts approachingthe theoretical values present (Table 1). Whenthese substances were hydrolyzed by acid for 2h and measured by the Warren assay (20) to see

how much TBARS was released, amounts weresomewhat less than the totals measured asabove. When the substrates were incubated withthe streptococcal supernatant 337 or the Vibrioenzyme, less sialic acid was released from BSMand fetuin by the streptococcal enzyme than bythe Vibrio enzyme, whereas more was releasedfrom IgG and human serum by the streptococcalenzyme.

Production of neuraminidase by streptococci.Of 19 T-type 12 strains recently isolated fromsore throats at Children's Memorial Hospital, 10produced neuraminidase in amounts that re-leased 1.47 to 5.09 ,ug of TBARS from BSM,with an average of 2.72 ,ug (Table 2). Five of theT-type 12 strains studied also were M-type 12,but none of these produced neuraminidase. Of11 T-type 4 strains studied, 10 produced neur-aminidase in slightly larger amounts which re-leased 1.73 to 6.89 ,ug of TBARS, with anaverage of 4.70 ,ug. None of the T-type 4 strainswas M-type 4. However, one was M-type 60 anddid produce neuraminidase. Only two of thestreptococcal supernatants that releasedTBARS from BSM also released it from fetuin;the amounts released from fetuin were smaller

than those released from BSM (1.08 and 2.1 ,ugfrom fetuin and 2.1 and 6.7 ,ug from BSM,respectively). In contrast, the Vibrio enzymereleased 17.7 ,ig of TBARS from BSM and 22.0,ug of TBARS from fetuin. None of the otherstrains from Children's Memorial Hospital pro-duced neuraminidase in our assays, although allof the 12 T-type 3 strains, 6 of the 11 T-type 1strains, and all of the 6 T-type 6 strains were M-typable. None of the five strains recently isolat-ed from patients with AGN (M-type 12 strains)or their siblings (T-type 9 strains) in Chicagoproduced neuraminidase. However, the stockM-type 12 strain isolated by Griffith more than25 years ago (SF42) which had lost its group Acarbohydrate during mouse passage and hadbeen frozen and lyophilized many times pro-duced enough neuraminidase to release 2.78 ,ugof TBARS.None of the 18 strains from patients with

AGN in Trinidad produced neuraminidase, al-though most were M-typable. These strains in-cluded M-types 55, 57, 60, 1, and 49. None wereT-type 12, but the M-type 60 was T-type 4. Ofthe 14 strains from patients with acute rheumaticfever in Trinidad, 2 produced small amounts ofneuraminidase; one group A strain which wasnot T-typable released 1.5 ,ug of TBARS, andone group G strain released 3.5 ,ug. None of thestrains from patients with sore throats in Trini-dad produced neuraminidase, and these includ-ed three T-type 12 strains (non M-typable). NoT-type 4 strains were in this group.

Direct evidence of neuraminidase activity inserum samples. When BSM was incubated withand without the sera from patients and with andwithout control sera and assayed for TBARS(20), the readings at 549 jiM and the amounts of

TABLE 1. Amounts of sialic acid or TBARS in substrates and freed from substrates by acid hydrolysis,streptococcal supernatant 337, and Vibrio cholerae neuraminidase

Amt (.Lg/0.1 ml) of SA' or TBARS in:Sialicacidmeasurement 1. P0% 0.5% Whole

BSM Fetuin 1.0% IgG 2.0% IgG serumb

Total Theoretical 50.0 33.0 2.34 4.20 60.0

Total measuredc Jourdian 44.0 34.0 1.32 2.27 61.0Svennerholm 61.0 32.0 1.61 58.0

Freed by acid hydrolysis Warren 42.0 24.0 0.93 1.85 45.0

Freed by streptococcal Warren 6.8 (16)d 12.6 (52) 0.77 (83) 1.70 (92) 44.0 (97)supernatant

Freed by Vibrio enzyme Warren 17.7 (42) 22.0 (92) 0.31 (33) 1.33 (72) 41.2 (91)a SA, Sialic acid.b A normal serum.c Means of two to four assays.d Percentage of that freed by acid hydrolysis is shown in parentheses.

INFECT. IMMUN .

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from

STREPTOCOCCAL NEURAMINIDASE AND NEPHRITIS 1199

TABLE 2. In vitro production of neuraminidase by 23 of 155 streptococcal strains testedSource of Associated T-type No. tested' No. producing TBARSstrains disease neuraminidase released (pLg)

Chicago Sore throat T-12 19 (5 M-12) 10 (0 M-12) 2.72bT-4 11 (1 M-60) 10 (1 M-60) 4.70T-3 12 (12 M-3) 0T-1 11 (6 M-1) 0T-6 6 (6 M-6) 0Others 34 0

AGN T-12 3 (3 M-12) 0

AGN (sibling) T-9 2 0

Stock SF42 Sore throat T-12 1 (M-12) 1 (M-12) 2.78

Trinidad AGN Several' 18 (1 M-60) 0

ARFd (patientor sibling) NPe 5 1 1.50

Group G 1 1 3.50Others 8 0

Sore throat Several 24 (3 T-12) 0

aNumber also M-typable is given in parentheses.b Mean amount of TBARS released by 0.1 ml of supernatant, 2.72 p.g.I None of these was T-12, whereas one was T-4 (M-60).d ARF, Acute rheumatic fever.e NP, No pool (not T-typable).

TBARS calculated by Warren equations 1 and 2were less in the tubes with sera than in thosewithout sera (Table 3). When fetuin was incubat-ed with and without the sera from patients andwith and without control sera, the amounts ofTBARS were greater in the tubes with sera thanin those without sera. However, the amounts ofTBARS in the tubes with sera and fetuin wereless overall than the amounts in tubes containingserum plus buffer. Therefore, we found no directevidence of neuraminidase activity in the seratested in these assays.

Indirect evidence of neuraminidase activity.Total and bound amounts of sialic acid (Table 4)calculated by the assay of Jourdian et al. (7)were increased rather than decreased in the serafrom AGN patients (mean, 103 ,xg/0.1 ml) ascompared with those from control subjects(mean, 76 ,ug/0.1 ml). However, these amountswere greatest in the sera from rheumatic feverpatients (mean, 166 ,ug/0.1 ml). Free sialic acid,calculated from the Jourdian assays of total andbound sialic acid, was present in small quantitiesin sera from two patients with AGN, threepatients with rheumatic fever, and one controlsubject. Total amounts of sialic acid obtained forsome of these sera by the method of Svenner-holm (19) were less than those obtained by the

method of Jourdian et al., but they were compa-rably higher and lower.Antibody to streptococcal neuraminidase (Ta-

ble 5) was not evident in any of the sera frompatients with AGN, since amounts of TBARSfound after incubation of these sera with thestreptococcal neuraminidase, followed by addi-tion of BSM or fetuin, resulted in increasedamounts of TBARS as compared with theamounts found after the enzyme was incubatedwith the substrate in the absence of sera. Al-though this was also true when rheumatic feveror control sera were incubated with the enzymeand followed by addition of BSM, TBARS wasdecreased after the addition of fetuin. This sug-gests some inhibition of the enzyme by therheumatic fever and control sera, in contrast tothe sera from patients with AGN. Moreover, innone of the assays did the TBARS in tubes withserum, enzyme, and substrate equal the sum ofTBARS in tubes with enzyme and substrate plusthat in tubes with serum and enzyme.

DISCUSSIONThe production of neuraminidase by strepto-

coccal strains from Chicago followed the patternthat others have reported (1) in that T-type 12and T-type 4 strains were the primary produc-

VOL. 38, 1982

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from

TABLE 3. Direct evidence of neuraminidase: release of TBARS from BSM or fetuin by sera from patientswith AGN or rheumatic fever and from control subjects

Amt (ILg) of TBARS ina:Serum BSM + Fetuin + Buffer +

Serum Buffer' Serumb Buffer' sem

Acute nephritis1 1.6 1.6 1.5 0.2 2.02 1.3 1.6 1.5 0.2 1.83 1.0 1.7 1.0 0.4 1.84 1.1 1.7 1.2 0.4 1.45 1.0 2.4 0.8 0.2 1.66 1.2 2.4 1.8 0.2 1.6

Rheumatic fever1 1.7 2.4 2.0 0.2 2.02 1.6 1.6 1.6 0.2 2.03 1.6 1.6 1.5 0.4 2.04 1.0 1.7 1.4 0.4 1.65 1.4 1.7 1.4 0.2 1.86 1.4 2.4 1.9 0.2 1.4

Control1 1.1 1.6 1.1 0.2 1.42 1.2 1.6 1.3 0.2 1.83 1.0 1.7 1.0 0.4 1.64 0.9 1.7 0.7 0.4 1.65 1.0 2.4 1.7 0.2 1.46 1.2 2.4 1.9 0.2 1.4a Amount of TBARS was determined by the method of Warren (20).bTubes contained 0.1 ml of serum and 0.1 ml of BSM, fetuin, or buffer.c Tubes contained 0.1 ml of BSM or fetuin and 0.1 ml of buffer.

ers. However, the results failed to support thehypothesis that neuraminidase production bynephritogenic streptococci is basic to the patho-genesis ofAGN, since none of the strains associ-ated with AGN in Chicago or Trinidad producedneuraminidase. Rather, two rheumatic feverstrains from Trinidad produced small quantitiesof this enzyme, as did the M-type 12 strain(SF42) isolated from a patient with scarlet fevermany years ago. These results were disappoint-ing and may be partly due to the length ofstorage of the nephritogenic strains from Trini-dad. However, the rheumatic fever strains hadbeen frozen for equal periods of time, and theSF42 strain had been lyophilized, frozen, andsubcultured repeatedly for more than 25 years.In addition, three type 12 strains freshly isolatedfrom patients with AGN in Chicago failed toproduce neuraminidase. It should be noted that23 streptococcal supernatants released TBARSfrom BSM, whereas only two of these released itfrom fetuin. Thus, although the fetuin was acleaner substrate with lower readings at 549 and532 ,uM than BSM, the latter was employed aswell as fetuin in all of our tests for streptococcalneuraminidase activity.

Neither direct nor indirect evidence wasfound for neuraminidase activity in the sera of

nephritis patients, in contrast to the sera ofrheumatic fever patients or control subjects.Rather, the assays of total and bound sialic acidwere consistent with the increased serum immu-noglobulins and other acute-phase reactants inthe sera of rheumatic fever patients and thelesser increase of these factors in the sera ofAGN patients previously found in Trinidad (13,15). Minimal amounts of free sialic acid werefound in the sera of three rheumatic fever pa-tients and one control subject and in the sera oftwo AGN patients. Evidence of antibodies toneuraminidase was absent from the sera of pa-tients with AGN. However, a decrease wasnoted in the TBARS released after incubation ofstreptococcal neuraminidase with sera from pa-tients with rheumatic fever or control subjectsbefore the addition of fetuin, but not of BSM.Thus, these observations do not support the

hypothesis of McIntosh et al. (8) and Rodriguez-Iturbe et al. (17) that streptococcal neuramini-dase degrades immunoglobulins so that theybecome autoantigenic and are the basis of neph-rotoxic immune complexes. The nephritogenicstrains of streptococci did not produce neur-aminidase, nor did the sera from patients withAGN show direct or indirect evidence of neur-aminidase activity. In contrast, Rodriguez-

1200 POTTER ET AL. INFECT. IMMUN.

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from

STREPTOCOCCAL NEURAMINIDASE AND NEPHRITIS 1201

TABLE 4. Indirect evidence of neuraminidase: totaland bound sialic acid in sera from patients with AGN

or rheumatic fever and from control subjectsAmt (4g/0.1 ml) of SAM obtained by

following assay:Serum Jourdian Svennerholm

Total - Bound= Free Total

Acute nephritis1 100 92 82 105 100 5 72.73 84 84 0 68.44 80 80 05 115 116 06 136 136 0

Rheumatic fever1 160 143 17 98.82 176 173 3 101.73 1% 190 64 200 200 05 110 110 06 154 156 0

Control1 61 62 0 50.02b 100 89 11 70.03 80 80 04 65 65 05 76 75 16 72 72 0a SA, Sialic acid.b A healthy, muscular young man in Chicago.

Iturbe et al. (16) found direct evidence of neur-aminidase activity in the sera of 8 of 39 patientswith AGN, 5 of whom still had active infections.Thus, it may be that by the time nephritis haddeveloped in our patients, neuraminidase was nolonger present, although it had been active dur-ing prior streptococcal infections. However, ourserum samples were obtained as soon as patientswere admitted to the hospital. Perhaps it is moresurprising that Rodriguez-Iturbe et al. (16) foundsuch evidence than that we did not, since at least5 days would be needed for production of anti-

bodies to the degraded immunoglobulin previ-ously present according to this hypothesis, andprobably more time would be needed for thedevelopment of immune complexes and glomer-ulonephritis. Furthermore, Gregoriadis et al. (4)found that most of a Clostridium neuraminidasehad disappeared from the serum within 5 h of itsinjection into rats. By the time AGN develops,therefore, all direct evidence of neuraminidase islikely to be gone. Even so, the indirect evidenceof decreased bound and increased free sialic acidcould have been present briefly and well beforethe patients presented with AGN. In contrast, itmay have been too early in the course of thedisease to demonstrate free serum antibody tostreptococcal neuraminidase. Such thoughtssuggest that direct evidence for neuraminidaseshould be sought in sera from patients withactive T-type 12 or T-type 4 infections instead ofthose with AGN, whereas antibodies to strepto-coccal neuraminidase might better be soughtlater in the course of poststreptococcal AGN.However, this relationship of neuraminidase tonephritis seems unlikely, since none of our

nephritogenic strains produced the enzyme. Theimportant observation by Fillit et al. (3) thatTBARS with highest reading at 532 FjM (which isnot sialic acid or deoxyribose and is dialyzable)is increased in patients with increased serumcreatinine does not apply to our patients, sinceserum creatinines were less than 2 mg/dl in all ofthe five sera with any evidence of TBARS.Furthermore, the observations by Harvey et al.(6) of increased glycoproteins and sialic acidlevels in the sera of patients with cancer warnsus of yet other possible causes for positive testsfor sialic acid which are not associated withstreptococcal infection.

ACKNOWLEDGMENTS

We thank Elizabeth Quamina of the Ministry of Health ofTrinidad and Tobago for permission to publish this report andJaglal Bhikie and Steve Reid for technical assistance.

This work was supported by Public Health Service grant AI

TABLE 5. Indirect evidence of neuraminidase: antibody to streptococcal neuraminidase 337 in sera frompatients with AGN or rheumatic fever and from control subjects

Mean amt (,ug/0.1 ml) of TBARS released in tubes containing:

Serawfrom Serum + Buffer + Serum + Serum + Serum + Serum +patients with: neuraminidase + neuraminidase + neuraminidase + neuraminidase + neuraminidase + neuraminidase +

BSMb BSMb buffer fetuin fetuin buffer

Acute nephritis 11.0 4.8 12.1 15.0 12.6 11.8Rheumatic fever 9.3 4.8 10.4 11.7 12.6 10.0Control subjects 7.9 4.8 8.6 11.2 12.6 8.8

a Six sera in each group.b Mean of three assays as sera from two nephritis patients, two rheumatic fever patients, and two control

subjects were run in each assay with these tubes without serum.

VOL. 38, 1982

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from

1202 POTTER ET AL.

14361 from the National Institutes of Health and by the OthoS. A. Sprague Memorial Institute.

LITERATURE CITED

1. Davis, L., M. M. Baig, and E. M. Ayoub. 1979. Propertiesof extracellular neuraminidase produced by group Astreptococcus. Infect. Immun. 24:780-786.

2. Ferrieri, P. 1975. Acute post-streptococcal glomerulone-phritis and its relationship to the epidemiology of strepto-coccal infections. Minn. Med. 58:598-602.

3. FilIt, H., E. Elion, J. Sullivan, R. Sherman, and J. B.Zabriskie. 1981. Thiobarbituric acid reactive material inuremic blood. Nephron 29:40-43.

4. Gregoriadis, G., D. Putnam, L. Louis, and D. Neerunjun.1974. Comparative effect and fate of non-entrapped andliposome-entrapped neuraminidase injected into rats. Bio-chem. J. 140:323-330.

5. Gutteridge, J., and T. Tickner. 1978. The characterizationof thiobarbituric acid reactivity in human plasma andurine. Anal. Biochem. 91:250-257.

6. Harvey, H. A., A. Lipton, D. White, and E. Davidson.1981. Glycoproteins and human cancer. II. Correlationbetween circulating level and disease states. Cancer47:324-327.

7. Jourdian, G. W., D. Lawrence, and S. Roseman. 1971. Thesialic acids. XI. A periodate-resorcinol method for thequantitative estimation of free sialic acids and their glyco-sides. J. Biol. Chem. 246:430-435.

8. McIntosh, R. M., D. B. Kaufman, J. R. McIntosh, and W.Griswold. 1972. Glomerular lesions produced in rabbits byautologous serum and autologous IgG modified by treat-ment with a culture of ,B-haemolytic streptococci. J. Med.Microbiol. 5:1-7.

9. Moody, M. D., J. Padula, and D. Lizana. 1965. Epidemio-logical characterization of group A streptococci by T-agglutination and M-precipitation tests in the PublicHealth Laboratory. Health Lab. Sci. 2:149-162.

10. Noble, R. C., and K. L. Vosti. 1973. Biologic and immu-nologic comparison of nephritogenic and nonnephrito-genic strains of group A, M-type 12 streptococci. J.Infect. Dis. 128:761-768.

11. Peller, 0. G. 1981. Neuraminidase and free sialic acidlevels in acute poststreptococcal glomerulonephritis. N.Engi. J. Med. 305:1016-1017.

12. Potter, E. V., and A. F. Moran. 1979. Extracellular fac-tors, blood group antigens, and bacteriophage of nephrito-genic and nonnephritogenic strains of M-type 12 strepto-cocci. J. Infect. Dis. 140:392-396.

13. Potter, E. V., M. A. Shaughnessy, T. Poon-King, andD. P. Earle. 1982. Serum immunoglobulin A and antibodyto M-associated protein in patients with acute glomerulo-nephritis or rheumatic fever. Infect. Immun. 37:227-234.

14. Potter, E. V., G. H. Stollerman, and A. C. Siegel. 1962.Recall of type specific antibodies in man by injections ofstreptococcal cell walls. J. Clin. Invest. 41:301-310.

15. Potter, E. V., M. Svartman, I. Mohammed, R. Cox, T.Poon-King, and D. P. Earle. 1978. Tropical acute rheu-matic fever and associated streptococcal infections com-pared with concurrent acute glomerulonephritis. J. Pe-diatr. 92:325-333.

16. Rodriguez-Iturbe, B., V. N. Katujar, and J. Coello. 1981.Neuraminidase activity and free sialic acid levels in theserum of patients with acute poststreptococcal glomerulo-nephritis. N. Engi. J. Med. 304:1506-1510.

17. Rodriguez-Iturbe, B., D. Rabideau, R. Garcia, L. Rubio,and R. M. McIntosh. 1980. Characterization of theglomerular antibody in acute poststreptococcal glomeru-lonephritis. Ann. Intern. Med. 92:478-481.

18. Swift, H. F., A. T. Wilson, and R. C. Lancefield. 1943.Typing group A hemolytic streptococci by M-precipitinreactions in capillary pipettes. J. Exp. Med. 78:127-133.

19. Svennerholm, L. 1957. Quantitative estimation of sialicacids. II. A colorimetric resorcinol-hydrochloric acidmethod. Biochim. Biophys. Acta 24:604-611.

20. Warren, L. 1959. The thiobarbituric acid assay of sialicacids. J. Biol. Chem. 234:1971-1976.

INFECT. IMMUN.

on January 8, 2019 by guesthttp://iai.asm

.org/D

ownloaded from