Salmonella typhi from Blood

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1978, p /78/ $02.00/0 Copyright 1978 Americn Society for Microbiology Lbortory nd Clinicl Investigtion of Recovery of Slmonell typhi from Blood K. C. WATSON Centrl Microbiologicl Lbortories, Western Generl Hospitl, Edinburgh, Scotlnd Received for publiction 16 August 1977 Vol. 7, No. 2 Printed in U.S.A. An experimentl study ws undertken of isoltion of Slmonell typhi from rtificilly infected whole blood nd blood clot prepred either with smll numbers of freshly isolted strins or with the S. typhi Ty2 strin. Results showed tht 8 ml of whole blood required minimum of 50 ml of bile slt broth to dilute the bctericidl effect of the serum. However, the isoltion rte for recovery in 210 cses of enteric fever ws only 64% when 50 ml of medium ws used, s compred to 92% recovery from blood clot from the sme group incubted in streptokinse bile slt broth. This probbly reflects very low grde bcteremi in mny of the ptients. Further investigtion showed tht, idelly, between 150 nd 200 ml of medium is required for stisfctory whole-blood culture. Both the experimentl study of rtificilly infected clots nd recovery rtes from ptients indicte tht rpid clot dissolution with streptokinse is preferble to whole-blood culture; this experience hs been mply confirmed by lmost 5,000 cses over 20-yer period. A 15-ml volume of bile slt broth with 100 U of streptokinse is dequte for the clot from 8 ml of blood, nd svings on medi nd incubtor spce hve considerble cost benefits in developing countries. Isoltion of Slmonell typhi from blood is the dignostic method of choice in typhoid fever. However, the reported incidence of positive isoltes vries enormously. In series in Delhi, Indi, only 527 isoltes were obtined from 5,735 suspected cses (9.2%) (5). This compres with 175 isoltes in 243 cses (72%) in Rhodesi (8) nd 219 isoltes from 298 clinicl cses in peditric group in Ntl (73%) (4). Our own experience, lso in Ntl, gve 86 isoltes in 147 ptients (58.5%) when whole blood ws cultured. Of these 147, 99 hd clot cultures performed, with 93 positive results (93%), fter removl of the serum nd dissolution of the clot with streptokinse (6). None hd positive blood cultures nd negtive clot cultures. The 54 ptients with negtive cultures, either blood or clot, were dignosed by unequivocl clinicl evidence of enteric fever nd confirmed by serologicl evidence of rising ntibody titers. Mny yers' experience of this lst technique using 15 ml of bile slt broth contining 100 U of streptokinse per ml hve confirmed the vlue of the method. In generl, physicl methods of clot lysis, e.g., glss beds nd metl filtrtion sieves, hve proved unstisfctory or dngerous in our experience. Even in severe bcteremic sttes, the number of orgnisms per milliliter of blood is smll, often less thn 10 orgnisms per ml (3, 7; K. C. Wtson, M.D. thesis, University of Aberdeen, Scotlnd). Since S. typhi is nonexcting in its culturl requirements, poor isoltion rtes stem either from indequte volumes of blood being smpled or from deth of orgnisms in culture bottles before growth occurs, due to indequte dilution of bctericidl serum fctors. An experimentl study ws crried out to determine the effect of medi nd blood volumes, to compre results of conventionl blood culture systems nd those from experimentlly infected blood clots, nd to consider the effect of serum on orgnism isoltion. The ppliction of whole-blood nd blood clot culture systems to isoltion of S. typhi from chnicl cses is lso discussed. MATERIALS AND METHODS S. typhi strins. Experiments were done either with strins of S. typhi freshly isolted from cses of typhoid fever in King Edwrd VIII Hospitl, Durbn, Ntl, or with the stock lbortory strin S. typhi Ty2 (NCTC 8385). Strins were mintined on nutrient gr slopes t room temperture. Culture medium. Stndrd 0.5% bile slt broth contining 5 g of sodium turocholte in 1 liter of sterile nutrient broth (ph 7.6) nd utoclved t 108 C for 15 min ws employed. Blood cultures were done by dding 8.0 ml of blood from ptients to vrying volumes of medium in 120-ml- sterile glss bottles. Continers were incubted t 37 C nd subcultured dily for 1 week onto Wilson nd Blir bismuth sulfite medium (9). The medium ws freshly prepred every 3 dys from solutions kept t 4.0 C, nd pltes 122

2 VOL. 7, 1978 RECOVERY OF S. TYPHI FROM BLOOD 123 were stored t this temperture. The solutions re stble for severl months. Negtive cultures were then subcultured weekly for 6 weeks before discrding. Isoltion of S. typhi ws crried out from blood clot s described before (6). Briefly, the clots from 5.0 to 8.0 ml of blood were dded fter retrction to 15 ml of bile slt broth contining 100 U of streptokinse (Vridse, Lederle, Americn Cynmid Co., N.Y.) dissolved in 0.5 ml of sterile 0.85% sline. This produces rpid lysis nd relese of trpped orgnisms. Streptokinse hs no hrmful effects on S. typhi. Subcultures were plted s bove. Preprtion of rtificilly infected clots. Overnight nutrient gr slope cultures of S. typhi were prepred by inoculting ech slope with four 0.02-ml volumes from 50-drop/ml pipette of n 8-h horse digest broth culture (1). Growth ws wshed off the slopes with sterile 0.85% sline contining 5% nutrient broth. Suitble 10-fold dilutions were then mde in the sme medium, nd plte counts were performed (2) Ḋilutions contining pproximtely 25 to 30 orgnisms per ml were pipetted in 1.0-ml volumes into sterile tubes (78 by 13 mm). Four milliliters of blood freshly drwn from volunteer ws then dded, nd the contents were mixed with gentle rottion until clotting occurred. The tubes were stood t 4.0 C for 2 h, nd the seprted serum ws removed. The clots were decnted into sterile petri dishes nd bisected by mens of two sterile microscope slides. A fresh plte nd slides were used for ech clot. Ech hlf clot ws then incubted s described below. Clotted specimens from ptients with clinicl enteric fever were delt with similrly. A series of preliminry quntittive experiments ws crried out to show tht the method of rtificil clot preprtion resulted in even distribution of orgnisms throughout the clots. This ws done by dividing clots into four equl prts, lysing with streptokinse, nd performing plte counts. Immune nd nonimmune ser. Immune ser were obtined from ptients in the convlescent phse of disese nd hd 0 nd H gglutinin titers greter thn 1,280. Nonimmune ser were obtined from norml helthy volunteers with no history of pst enteric infection or of immuniztion with vccine contining S. typhi, S. prtyphi A, or S. prtyphi B. 0 nd H titers were less thn 20. Het inctivtion of serum ws crried out t 56 C for 20 min. RESULTS Dilution effect in blood culture. Vrying volumes of fresh blood obtined from volunteers who hd previously been immunized with TAB (typhoid-prtyphoid A-prtyphoid B) vccine nd whose ser contined 0 nd H ntibodies to S. typhi were dded to 5.0-ml mounts of bile slt broth contining pproximtely 25 to 30 S. typhi orgnisms in sterile 28-ml glss screwcpped bottles nd incubted t 37 C. Dily subcultures were mde onto bismuth sulfite medium. Results re detiled in Tble 1 for one representtive experiment. Tble 1 clerly indictes the inhibiting effect of finl serum concentrtions greter thn bout 1/8. This confirms the need to ensure tht dequte volumes of medium re used in blood culture systems if negtive results re to be voided. Similr results were obtined when the volume of blood ws kept constnt nd the medium volume vried. When 8.0 ml of whole blood ws dded to 100, 75, or 50 ml of medium contining pproximtely 20 vible orgnisms, growth ws obtined on subculture fter 24 h. However, with medium volumes of 15, 25, nd 35 ml, no growth ws evident fter 14 dys of incubtion. Although these findings suggested tht 50 ml of medium ws dequte for 8 ml of blood, our comprtive findings for whole-blood culture nd clot culture in clinicl cses of enteric fever did not ber this out, presumbly becuse of very low degrees of bcteremi in some ptients (see below). Effect of clot lysis on isoltion of S. typhi. Tble 2 illustrtes the results obtined with 50 bisected clots, where one hlf of ech clot ws incubted in streptokinse bile slt broth nd the other hlf in bile slt broth lone. Two points re pprent from Tble 2. Of the 50 hlf clots, 16 (32%) incubted with bile slt broth lone filed to grow fter 14 dys of incubtion, nd 12 of the 34 positive cultures (35%) required 3 or more dys of incubtion before becoming positive. Where the corresponding hlves of the sme clots were lysed by streptokinse, ll 50 were positive within 48 h of incubtion. It is unlikely tht the negtive results were due to restricted distribution of orgnisms within the clots. Effect of immune nd noimmune ser on isoltion of S. typhi from rtificilly infected clots. Artificilly infected clots were prepred by dding 4.5 ml of blood from n immune or nonimmune donor to 0.5 ml of suspension of S. typhi contining pproximtely 30 orgnisms. After bisection of the clots, one hlf of ech ws dded to 10 ml of streptokinse bile slt broth long with either the totl volume of serum originlly present or n equivlent volume of sterile 30% bovine serum lbumin. Results fter incubtion for 14 dys re shown in Tble 3. In the experiment where the serum ws discrded, ll 28 clots mde from nonimmune donor grew S. typhi within 24 h (Tble 3). Where the serum cme from n immune donor, two clots were negtive fter 14 dys. We hve shown previously tht orgnisms trpped in clots my be killed off by the residul smll mounts of serum tht re still present nd which re not diluted out till lysis occurs (K. C. Wtson, M.D. thesis). Those hlf clots incubted with nonimmune serum yielded 17 positive cultures out of the 28 (60.7%), s compred with only 7 out of

3 124 WATSON TABLE 1. Effect of vrying blood volumes with constnt medium volume on recovery of S. typhi Approx Dys of incubtion Medium vol- Blood vol- plsm diluume (ml) ume (ml) tion : : : : : : : : S. typhi Ty2. Number of orgnisms per dilution, pproximtely 25. +, Positive isoltion; -,negtive isoltion. TABLE 2. Effect of streptokinse on recovery of S. typhi from rtificilly infected nd bisected blood clots Hlf clots in strepto- Hlf clots in bile broth N.ofinby kinse bile broth lone incub - tion Positive Negtive Positive Negtive S. typhi strin loclly isolted (no. 1321). Number of clots prepred, 50. -, Not cultured on this dy. 51 (13.7%) where clots were prepred from n immune donor nd incubted with serum; these results exemplify the bctericidl effect of ntibody nd complement on grm-negtive orgnisms. Effect of fresh nd heted immune serum on S. typhi. Five-milliliter infected clots were prepred s bove, using n immune donor. Serum ws removed fter 2 h t 4.0 C from hlf the infected clots nd het inctivted s described. Ech clot ws then bisected. Hlf of ech ws incubted in 15 ml of streptokinse bile slt broth. The other hlf ws incubted long with ll the serum in 10 ml of streptokinse bile slt broth, nd subcultures were mde s before. Results showed tht of the 16 hlf clots incubted with inctivted serum, 14 were positive fter 24 h of incubtion, nd 2 were still negtive fter 14 dys. However, of the 16 incubted with fresh serum only one gve positive result fter 14 dys of incubtion. All 32 hlf clots incubted without serum gve positive cultures fter 1 dy of incubtion. Isoltion of slmonelie from ptients with clinicl enteric fever. Concurrently with these lbortory studies, the isoltion of slmonelle from ptients with clinicl enteric fever ws studied. In series of 152 ptients with J. CLIN. MICROBIOL. TABLE 3. Effect of immune nd nonimmune serum on isoltion of S. typhi from rtificilly infected blood clots Culture results with: No. of -- Serum dys of Serum present Serum removed incubtion Positive Neg- Positive Negtive tive Immune Nonimmune Approxiimtely 30 orgnismns per clot. Clots incubted in streptokinse bile slt broth. S. typhi strin loclly isolted (no. 386). -, Not subcultured on this dy. unequivocl clinicl enteric fever seen during the first 10 dys of illness, the residul clots obtined from 8.0-rnl volumes of blood were cultured fter discrding the serum. Of 48 incubted in bile slt broth, 36 were positive (75%) fter 21 dys of incubtion. Of these, 25% were positive fter 1 dy of incubtion, nd 45% were positive fter 2 dys. Of the remining 104 incubted in streptokinse bile slt broth, 98 (94%) were positive t 21 dys. Of these, 52% were positive fter 1 dy nd 85% were positive fter 2 dys of incubtion. These findings re detiled more clerly in series of 18 ptients, where the blood clots were bisected, hlf were incubted in bile slt broth lone, nd the other hlf were incubted in streptokinse bile slt broth (Tble 4). All of the 18 hlf clots in streptokinse medium were positive nd ll except one were positive within 72 h. However, one-third of the corresponding hlf clots incubted in bile slt broth lone filed to grow, nd three of them required 5 to 8 dys to become positive. Medium volume in blood cultures from

4 VOL. 7, 1978 RECOVERY OF S. TYPHI FROM BLOOD 125 ptients. Two volumes of bile slt broth were compred, 35 ml nd 50 ml, following the experimentl findings tht 50 ml of medium might be dequte for whole-blood cultures. Blood volumes of 8.0 ml from clinicl ptients were cultured. The first series contined 104 ptients nd the second contined 210. At the sme time, replicte 8.0-ml volumes were collected for clot culture in streptokinse broth. Results re detiled in Tble 5. The percent isoltion rte for whole-blood culture incresed from 50 to 64% when the medium volume ws incresed from 35 to 50 ml, nd the men time of incubtion required for positive result dropped from 4.8 to 2.0 dys. This compres with 93% isoltion rte nd men incubtion time of 1.9 dys for ll 314 ptients TABLE 4. Culture results from bisected blood clots in enteric fever Dys of incubtion for positive isoltion in: Ptient no. Orgnism Streptoki- Bile broth nse broth S. typhi S. typhi S. prtyphi A S. typhi 5 1 neg. t 14 S. typhi S. typhi S. typhi S. typhi S. typhi 10 2 neg. t 14 S. typhi 11 2 neg. t 14 S. typhi S. typhi S. prtyphi A S. typhi 15 3 neg. t 14 S. typhi 16 3 neg. t 14 S. typhi 17 3 neg. t 14 S. typhi S. typhi Percent positive Hlf of ech clot ws incubted in bile broth nd hlf ws incubted in streptokinse bile broth. TABLE 5. when similr volume of clotted blood ws cultured. DISCUSSION Aprt from suitble culture medium, there re two essentil requirements for isoltion of slmonelle from blood. These include n optimum smple volume to tke ccount of the low level of bcteremi nd method tht will prevent killing of orgnisms by serum fctors, especilly ntibody nd complement. In generl, smple volume of 8 to 10 ml is lmost lwys dequte. Few negtive results re to be expected from rndom distribution of orgnisms when this is done. If whole blood is to be cultured, it is essentil to prevent bctericidl serum effects either by dequte dilution of the smple in n dequte medium volume or by inhibition of serum bctericidl fctors. Poor culturl results quoted in the literture pper to be lmost entirely due to filure to recognize tht rpid killing of smll numbers of bcteri will occur unless this is done. This pplies prticulrly where ptients re first seen during the second nd subsequent weeks of illness, when ntibody is being freshly produced. The ddition of complement-destroying gents such s sodium polynetholsulfonte (Liquoid) my be helpful, but not ll orgnism killing is due to ntibody- nd complement-medited mechnisms. The comprtive results in Tble 5 indicte tht 50 ml of medium with 8 ml of blood is indequte for whole-blood cultures. Our own further investigtions hve shown tht somewhere in excess of 100 ml nd preferbly nerer 200 ml of medium is needed for 8 ml of blood if comprble isoltion rtes re to be obtined for blood clot nd whole-blood cultures. In series of studies of 100, 150, nd 200 ml of medium for 8 ml of blood for whole-blood cultures, the isoltion rtes compred to those from blood clot were 86, 94, nd 100%, respectively. However, Comprison of 35- nd 50-ml volumes of culture medium for whole-blood cultures (8 ml of blood) nd isoltion from blood clot in clinicl cses of enteric fever Avg time of incu- No. of p- Culture nd medium Voliofme- Positive iso- Negtive iso- for %positivebtion positive tients d(ml ltion ltion (ml) ~~~~~~~~~~~~~(dys) ml of whole blood in BSB Blood clot from 8 ml of blood in SKBSB ml of whole blood in BSB Blood clot from 8 ml of blood in SKBSB BSB, Bile slt broth; SKBSB, streptokinse bile slt broth.

5 126 WATSON such lrge volumes my present problems, especilly in developing res where the disese is common, in terms of cost of medium nd necessry incubtor fcilities to del with lrge numbers of cultures. Isoltion from blood clot is fr more relible, provided rpid lysis occurs, since orgnisms trpped within the clot re susceptible to serum fctors dsorbed to the fibrin meshwork (K. C. Wtson, M.D. thesis). We hve utilized the method of lysis with streptokinse in bile slt broth in series of lmost 5,000 cses of enteric fever over period of 20 yers nd hve been impressed by the high isoltion rte. As shown in Tble 5, this is of the order of 92 to 94%. The fcility of using only 15 ml of medium for blood clot culture from 8 ml of blood is n enormous sving. We hve lso found tht it is esy to spot-inoculte 25 subcultures onto one petri plte of Wilson nd Blir bismuth sulfite medium by using n illuminted ruled templte. We recommend tht the clot technique hs mny dvntges over conventionl wholeblood culture, both in relibility nd in cost. J. CLIN. MICROBIOL. LlTERATURE CITED 1. Cruickshnk, R., J. P. Duguid, B. P. Mrmion, nd R. IL A. Swin (ed.) Medicl Microbiology, 12th ed., p Churchill Livingstone, Edinburgh. 2. Miles, A. A., S. S. Misr, nd J. 0. Irwin Estimtion of bctericidl power of blood. J. Hyg. 38: Pulvertft, R. J. V Bcteril blood cultures. Lncet i: Scrgg, J., C. Rubidge, nd H. L. Wllce Typhoid fever in Africn nd Indin children in Durbn. Arch. Dis. Child. 44: Sen, R., nd S. N. Sxen Typhoid fever in Delhi re: n ssessment bsed on bcteriologicl nd some epidemiologicl findings. J. Indin Med. Assoc. 50: Wtson, K. C Clot culture in typhiod fever. J. Clin. Pthol. 7: Werner, A. S., C. G. Cobbs, D. Kye, nd E. W. Hook Studies on the bcteremi of endocrditis. J. Am. Med. Assoc. 202: Wicks, A. C., G. S. Holmes, nd L. Dvidson Endemic typhoid fever. A dignostic pitfll. Q. J. Med. 40: Wilson, W. J., nd E. M. M. Blir Use of glucose bismuth sulphite iron medium for isoltion of B. typhosus nd B. proteus. J. Hyg. 26: Downloded from on October 20, 2018 by guest