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Studies on the virulence of Diphtheria bacilli Bain, Janet Burnett 1924

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STUDIES ON THE VIRULENCE OF DIPHTHERIA BACILLI. by J . B . Ba in , B.A. A p r i l 2 5 t h , 1924. STUDIES OH THE VIRULENCE OP DIPHTHERIA BACILLI, by Janet Burnett Bain, B. A. A Thesis submitted for the Degree of MASTER OF ARTS in the Department of BACTERIOLOGY. THE UNIVERSITY OP BRITISH COLUMBIA, APRIL, 1924. -1-STUDIB3 OH THE 7IRPLEH0B OP DIPHTHERIA BACILLI. laVroflttoUoa. It has been the custom, until recent investi-gation has established the value of the intracutaneous virulence test, to release individuals from quarantine for diphtheria after two successive negative nose and throat cultures, at least 24 hours apart, have been ob-tained; that is, quarantine has been based on morpholog-ical rather than biological characteristics. A large amount of work has been done which proves that there is considerable variation in the virulence of different •trains of diphtheria bacilli and that there are strains which are morphologically identical with virulent diph-theria bacilli which prove to be non-virulent when tested by animal inoculation. The danger to the community lies not in the presence alone of diphtheria bacilli but of organisms which are virulent. For this reason, a deter-mination of virulence, especially in non-clinical oases, seems to provide a reasonable means, of decreasing the quarantine in such oases. The present investigation has been undertaken to determine the virulence of routine diagnostic cultures, in clinical cases, contact and carriers, as are found in school room epidemics. The investigation oovered the -2-period from June 1st, 1923, to December 31st, 1923, dur-ing which all cultures received at the Laboratories of the Vancouver General Hospital, showing diphtheria bac-illi microscopically, were examined as to virulence. It is of interest at this point to review some of the work that has been done in connection with the virulence of diphtheria bacilli. According to Nuttall and Graham-Smith (1), "a correct estimate may be formed of the virulence of the bacilli toward guinea pigs and satisfactory evidence is yet lacking to show that races non-virulent to guinea-pigs can produce serious disease in man. She results of those experiments which have been made under uniform conditions.... show that the very great majority of raoes of diphtheria baoilli are either fully virulent or totally non-virulent for these animals.1* Subcutaneous virulence tests were used for many years with successful results but this method entails the loss of much time and considerable expense in laboratory animals. The standardization of diphtheria toxin by in-tracutaneous inoculation suggested a method of testing the virulence of diphtheria bacilli. Nelsser (2) com-pared the intracutaneous method with the subcutaneous and obtained results identical in both oases, Kolmer and Moshage (3), on the other hand, obtained better re-sults by subcutaneous inoculation, securing 86.5 % -3-positive results by subcutaneous inoculation and 64.9$ positive results by intracutaneous inoculation of the same cultures. Weaver (4) found no discrepancy between subcutaneous and intracutaneous reactions of 26 pure cultures of diphtheria bacilli. Recently, Horace It. Powell (5) compared the two methods and obtained ident-ical results in every case. The coexistence of virulent and avirulent organisms has been commented on by Wade and Vaughan (6), Nuttall and Graham-Smith (1), Kolmer and Uoshage (3) and others. This fact introduces a source of error in using pure cultures only as there is no apparent difference in appearance between colonies of virulent and avirulent organisms. With these facts in mind, Force and Beattle (7) used the "whole culture" method of testing virulence, suggested by Wayson, which consists of the intracutaneous inoculation of a saline suspension of field cultures. XftlfeO&s. The methods used in th is i n v e s t i g a t i o n are s i m i -lar to those employed by Force and B e a t t i e . All t e s t s were made from routine d iagnos t i c nose and throat c u l -tures on L o e f f l e r ' s blood serum s l a n t s , which had been incubated for 18 hours at 37^° C. The d iagnos i s was based on Wesbrook's (8) c l a s s i f i c a t i o n , granular types A, 0, and D only, being recognised. A l b e r t ' s (9) s t a i n -4-was used exclusively throughout In staining the prepar-ations for diagnosis. Attempts were made to isolate pure cultures from each culture with which a field culture test was made. This procedure consisted of transferring a loopful of the field culture by a platinum loop to another tube of Loeffler's blood serum slant, and making successive trans-fers on six tubes of media. After 18 to 34 hours incub-ation at 37£° 0, well isolated colonies were picked and streaked crosswise on blood serum slants, about 4 oross-streaks to a tube. She streaks were examined the follow-ing day and if pure were transferred to a separate tube of serum. If no pure cultures were obtained more colonies were picked and successive streaks of the original field culture were made again and the process repeated until pure cultures were successfully isolated or until it was considered impossible to isolate the diphtheria organisms. Two to three cc. of physiological salt solution were added to the blood serum slant and the growth scraped off with a platinum loop. The suspension was then transferred to another tube and diluted with salt solution to 8 to 10 cc., aooording to the turbidity. Lighter suspensions were made of pure cultures. Guinea pigs were prepared by shaving the abdomen and injected intracutaneously with a sufficient amount to produce a bleb approximately 10 mm. in diameter. Pour to six tests -5-were made on each pig. Duplicate pigs were used in every case, the control pig receiving 500 units of antitoxin subcutaneously before injection. After the death of several of the test pigs, it was decided to protect them by injecting 500 to 1000 units of antitoxin after 24 hours, The results obtained after this procedure was adopted were more satisfactory. Observations were made at the end of 24 hours, 48 hours and 72 hours. Force and Seattle (7) describe the appearance of the lesion as a "reddening and induration followed by a necrosis which manifested Itself by a white area with an extending pur-ple center, the "target stage", terminating in sloughing of the epidermis and crust formation. The protected animals, inoculated with virulent field cultures showed marked redness and induration of less diameter than In normal animals. In some instances a central brown crust appeared at the end of 48 hours, after which redness and induration rapidly disappeared. This brown crust in no way resembled the white and purple concentric area de-scribed above and could not be confused with it unless lesions were not observed before the third day." lejtal^g. In all, 250 cultures were investigated, the de-tails of which are recorded in the Appendix. Field cul-tures were tested in every instance and pure cultures -6-wherever it was found possible to isolate. We failed to isolate pure cultures in 101 out of 250 cultures* or 40.4 per cent. In the field cultures corresponding to these, 27 proved to be virulent and 74 non-virulent. She 27 virulent cultures were obtained from 17 clinical oases, 3 contacts, 5 carriers, 1 scarlet fever case and 1 whose source is undetermined. Failure to isolate such a large percentage was due in part to the presence in the mixed culture of spore bearering and other organisms which rapidly outgrew the diphtheria bacilli and in part to the paucity of diphtheria organisms in many of the cultures. Of the 101 cultures of which no pure cultures were isolated, 26 were morphologically positive in the nose culture only and 24 were from combined nose and throat swabbings of school room contacts or carriers, in many of which the smears show only a very small per-centage of diphtheria baoilli. There are, therefore,t149 cases in which field and pure cultures were tested. Of these, the pig died in 15 cases, which leaves 134 as a basis of comparison. Of this number the results coincided in 101 tests or 75.3 per cent. The field culture was positive and the pure culture negative in 11 or 8,2 per cent of the tests. The field culture was negative and the pure culture posi--7-tive in 22 or 16.4 per cent of the tests. The results recorded are at variance .vith those obtained by other workers; further work is being done to oheck our results. Bull and licKee (10) isolated pure strains from 167 cul-tures which gave negative results when tested for viru-lence by the field culture method. In not a single in-stance was a virulent strain isolated from these 167 cultures. Of 31 cases which gave positive reaotions in the field cultures, 26 virulent strains were isolated and they consider it probable that the other 5 cultures oontalned virulent organisms which escaped isolation. Havens and Powell (11) tested 295 cultures by both whole oulture and pure oulture methods, 275 (93 per cent) gave the same result by both methods. Of the 20 discrepancies, 8 were positive with whole oulture and negative with the pure culture and 12 were negative with whole culture and positive with the pure. These results Indicate that there may not be present in the field oulture a suffioient number of virulent diphtheria bacilli to give a charac-teristic lesion but when isolated, their virulence becomes apparent. The coexistence of virulent and non-virulent strains has been mentioned. Where the field oulture was positive and the pure oulture negative It is possible that the pure cultures were obtained from oolonies of non-virulent strains and the virulent strains missed. - 8 -gllalaal gja&t. Of the cultures examined, 128 were from "clinical" cases. The available information on the question as to whether they were actually clinical oases is open to some discussion. Undoubtedly some included under this cate-gory should properly be placed as "contact" or "carrier". Of such field cultures tested, 82 (65 per cent) gave posi-tive virulence; 37 (28 per cent) gave negative virulence and in 9 (7 per cent) the test animal died and the test could not be repeated. In these 128 cases, 92 pure cul-tures were isolated, of which 74 (60 per cent) gave posi-tive virulence and 18 (20 per cent) negative virulence. The 74 cases, however, constitute only 57 per cent of clinical cases, gaaiaaA* ana Carriers. The same difficulty exists here as with clinical eases. Accepting with reservations the classification as correct, these totalled 90. The field was positive in 26 oases (28,8 per cent); the field negative in 57 163.5 per cent); and the pig died in 7 (7,7 per cent). In these 90 cases, pure cultures were isolated in 39 instances, in 26 (66.6 per cent) of which virulence was positive and in 13 (33.3]}er cent) virulence was negative. ihe relatively high percentage of positive virulence is -9-probably explained by the fact that a great many of these were nasal cases, containing only a few B, diphtheriae and many contaminating spores and slimy organisms which made isolation impossible. It is probable that in many of these the virulence would have been negative had it been possible to isolate the diphtheria organisms. Kelly and Potter (12) compared the results of field culture virulence tests of oases and contacts and carriers in a general epidemic. Of 108 tests, 89 per cent of the cases gave a positive virulence and 11 per cent negative; 54 per cent of contacts and carriers gave positive virulence and 46 pet cent negative. In a school survey where diph-theria was prevalent but not epidemic, 25 per cent only gave positive virulence. 250 cultures have been studied. In this series there is a lack of exact oorelation between clinical symp-toms and field tests, a relatively high percentage of negative virulence being obtained. This is explainable in part by failure to obtain accurate data regarding the physical condition of the patient. There is a slight advantage in favor of the use of pure cultures, not recorded to as great an extent by other observers. This advantage, however, is more than -10-offset by tine sared in the use of field oultures and the inability to isolate pure cultures in many cases. 3erial Ho. 1 2 3 4 5 6 7 8 9 10 11 12 19 14 15 14 17 IS 19 20 21 22 29 24 25 24 27 26 29 80 21 32 89 84 35 36 37 38 39 40 41 42 48 44 *e 46 47 48 Hosp. Ho. 2384 2886 2889 289 6 2933 3014 3407 3460 3559 8489 8866 3572 3524 8654 3110 3124 8130 8133 8642 3582 8787 3841 3990 4018 4015 4486 4542 4594 4602 5052 6113 5212 5152 4946 5106 5247 5269 5300 5304 5310 5380 5382 5476 5515 5541 5541 6589 5600 APPBHDIX Source T T H H & T T T T H & * H & H k f H & H & N & H & H it H & T H H H H & H & H H H H H & H & H H « T T * •P H H & H & * T H H T T T T T T T * T T T * T T T T T I Physician' Diagnosis Olinioal if it n n H N M l» N Contact Olinioal Olinioal Oontaot * « Olinioal Carrier Olinioal Carrier N M Oontaot Olinioal n Carrier Olinioal M It H Oarrler N Olinioal M W H Oontaot Olinioal it M »t H It Carrier it • (S) is) (S) (S) ( 9) (s) (s) (3) (3) . (3) (sj Virnl Field • • 0 • • 0 • + • * • * + 0 • * + 0 0 • •+ * 0 • 0 0 0 0 • + 0 + • + 0 0 0 + * t 0 0 0 0 + • t 0 0 + enoe Pure * + 0 + • + + * • * • • + 0 • • • - • • * • • -* -0 • * • 0 • + 0 • + + -+ • + -0 + + + 0 Serial Ho. 49 50 51 52 53 54 55 56 57 56 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 78 76 77 78 79 80 81 82 83 84 85 86 87 68 89 90 91 92 95 94 95 96 HOBp. Ho. 5602 5757 5789 5833 6030 6117 6137 6294 6329 6332 6401 6345 6479 6482 6507 6509 6541 6553 6550 6590 6615 6727 6737 6806 6807 6835 6856 , 6670 6819 6968 7008 7011 6988 7018 7043 7044 7069 7075 7077 7082 7126 7163 7173 7191 7212 7216 Source T T T T T H I H T H & T H & T H H & T H & T * H H t H * T T H & T * H H & T N & T T H & T H & T H & T H & T H & T H & T N & T N & T H H H T T H H H & T H T Physician*s Tirulenoe Diagnosis Clinical Oarrier M M Clinical Contact Clinical M M It N Carrier w Clinioal Carrier M w Contact Clinioal n n M Snap. Bac Clinical Oontaot Clinical Carrier Clinical Clinical Carrier it Contact Oarrier Clinical N Oontaot Oarrier t» Clinical Carrier Scarlet Carrier Clinical Contact (3) IS) (S) (s) >.3) (3) (s) IS) (s) (s) (s) (s) (3) fever Field + p.d. p.d. 0 0 + 0 + * • 0 + 4 p.d. p.d. p.d. p.d. • 0 + • + * 0 + 0 • 0 + + • 0 + 0 4 0 0 0 0 0 4 4 0 4 0 + 0 0 0 Pure 4 4 0 • -4 -4 -* -4 4 4 * 0 4 4 0 0 4 0 4 4 -* 4 -0 4 4 * Serial H<U 97 9 8-99 100 101 102 193 104 105 106 107 108 109 110 111 112 118 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 150 151 132 133 134 135 136 137 138 139 140 141 142 143 144 Hosp. Ho. 7 237 7244 7281 7286 7245 729 6 7312 7338 7431 7378 X412 7471 7502 7522 7539 7600 7572 7610 7637 7735 7787 7805 7827 7830 7832 7975 8112 8126 8167 8199 8218 8220 8106 8265 8267 8268 8313 8315 8337 8344 8404 8440 8452 8459 8462 8463 8467 847 8 Source N T T N & T H T T H H & T H & T H & T T T H & T N & T T f * H & T H T T H T T I T T H ft T H & 7 H & T H & T H & T H H & T N & T T 5f H I H & 7 N & T H ft T H & J T H & T H T Physicans's Diagnosis Contact Clinical Carrier Clinioal Contact Clinical Carrier Contact Oontact (S) Susp. bao.S) " w (S) Carrier Clinical Carrier (3) is) Clinical n Carrier Clinical Carrier Clinical N tt Clinical n H H « (3) is) Contact 13} IS) Clinical n N M 11 Scarlet fever Clinioal Carrier «• Oontact Clinioal Contact H M Clinical Virulence Field 0 0 0 + e + 0 ••• 0 0 0 0 0 0 0 0 • 0 + p.d. + * t * * 0 + + + 0 0 0 0 0 0 0 + • 0 + 0 0 0 + 0 0 0 0 Pure 0 0 + • 0 + + • • -0 + • •*• + + 0 ' * + 0 0 + -• 0 + • * • -0 0 Serial Ho* 145 146 147 148 149 150 151 162 153 154 165 156 157 168 159 160 161 162 168 164 165 166 167 16$ 169 170 171 172 179 174 175 176 177 178 179 180 181 182 18S 164 185 186 187 188 189 190 191 Hoap. Ho. 849 0 8543 6550 8648 8697 6698 8739 8784 8811 8815 8851 8856 9007 9135 9151 89 02 8912 9216 9219 9 262 9288 9356 9360 9432 9502 9549 9566 9608 , 9 623 9637 9652 9661 9855 9861 9866 9753 9766 9798 9903 9911 9995 999 8 9 879 10061 10065 10066 10067 Source T H T H T H & T H & T H & T H & T H & I T H N & T H & T N & T N & T T H T N & T H & T I T N & T * H & T H & T I H & T H & T H & I T H & T N & T H & T T T H T T N & T H & T T T H & T H & T H & T Physician's Virulence Diagnosis Field Pure Clinical it it Contact Clinioal M Carrier tt Clinical n n Carrier « it n Clinioal Oarrier Clinical Carrier Clinical N N It Carrier Clinical Contact Clinical Carrier Clinical M Carrier H Clinioal Carrier Carrier Clinical M Carrier a Clinical H n M (3) IS) (3) (s) (3) (3) (3) (3) (3) (3) (8) (3) IS) (3) (3) 0 • • » • 0 + 0 0 0 -t 0 • 0 0 •t 0 0 0 0 0 • * • 0 • + + 0 * + 0 • •* t + • • 0 0 + + e 0 •• + • p.a. p.d. p.d. p.d. • • 0 • • * • • • 0 • 0 • • * • + * + •* * 0 + * * e S e r i a l Ho. 1 9 2 1 9 3 1 9 4 1 9 5 19 6 197 1 9 8 199 200 2 0 1 2 0 2 2 0 3 2 0 4 2 0 5 2 0 6 207 208 209 210 2 1 1 212 2 1 3 2 1 4 215 216 2 1 7 2 1 8 219 220 2 2 1 2 2 2 223 2 2 4 225 2 2 6 227 228 229 230 231 2 3 2 235 2 3 4 2 3 5 236 237 258 H o s p . Ho. 1 0 0 6 8 1 0 1 0 7 1 0 1 1 1 1 0 1 6 9 1 0 2 1 7 1 0 2 2 2 1 0 2 9 3 1 0 3 6 0 1 0 4 9 4 1 0 4 9 6 1 0 5 0 3 1 0 5 1 2 1 0 5 8 9 1 0 6 1 5 1 0 7 2 4 1 0 7 2 6 1 0 7 3 6 1 0 7 5 0 1 0 8 0 8 1 0 8 2 4 1 0 8 3 0 1 0 8 4 7 1 0 8 6 7 1 0 8 9 2 1 0 9 6 9 1 0 9 7 4 1 0 9 8 1 10996< 1 1 0 1 0 1 1 0 3 0 1 1 0 7 6 1 1 1 0 7 1 1 1 7 0 1 1 1 7 7 1 1 3 0 7 1 1 2 8 0 1 1 3 8 0 1 1 4 1 5 1 1 4 1 7 1 1 4 6 1 1 1 5 4 2 1 1 5 6 6 1 2 7 8 2 1 2 7 8 0 1 2 9 2 3 1 2 9 4 5 1 2 9 5 7 S o u r c e H & H & » & H I 'A1 H & H & H & T H T H I I T I T H & H & H H T - T S H & N & H & H & H & H & H T H & H & T T H & T I N & T T H & T H & T IT & T T T I T T T T S T T T 5? T •r T T T T T T I P h y s i c i a n ' D iagnos i s C l i n i c a l it it H C a r r i e r C l i n i c a l H Contact C l i n i c a l Carr i er C l i n i c a l »» It S (.81 S c a r l e t f e v e r C l i n i c a l n M It C a r r i e r Meas les C l i n i c a l « •» Contao t It it C l i n i c a l Contact C l i n i c a l Contac t Carr ier Contact C a r r i e r Contact C l i n i c a l C a r r i e r C l i n i c a l ( S ) is) (s) ( S ) ( 3 } (s) ( S ) ( 3 ) (SJ T o n s i l l i t i s C l i n i c a l M H II It It ( S ) ( S ) ( 3 ) V i r u l e n c e F i e l d P . d . 0 0 o • + 0 p . d . + + + 0 p . d . p . d . 0 • • + + 0 0 • * • • 0 +.• • 0 + 0 0 • • 0 0 0 0 0 0 0 0 p . d . p . d . + + + Pur 0 0 + -•* • * -0 + + 0 * * + 0 • + + + + + • + + + 0 • * • 0 0 + Serial Ho. 239 240 241 242 243 244 245 246 247 246 249 250 Hosp. No. 13097 13115 13190 13194 13195 13202 13208 13637 13710 13739 13736 13696 Source N N T I N T T T T & T H T & T T Physician' 8 Diagnosis Contact n Clinioal H Carrier Clinioal Carrier Clinical Contact Carrier Clinical Carrier (S) Virul Field p.d. p.d. + • « t 0 + + • * 0 enoe Pure + + + • + • + + + * + Source. N indicates a nose culture, 9 indicates a throat culture. I & I indicates a mixture of nose and throat cultures, either on one tube, as i s the routine method in school epidemics, indicated by the l e t t er S, under Physician's Diagnosis, or of separate positive nose and throat cultures . ?hyglciaft'g PAagnQglg. Clinical indicates the presence in the patient of signs of the disease s4ch as temperature, sore throat, membrane, adonitis , odor, and toxaemia. Carrier indicates an individual with no history of the disease or contact. Contact indicates recent close assoc-iation with c l in i ca l cases or carriers. The l e t t e r (S) indicates cases found in routine investigations of schools. Virulence. + indicates a typically positive intracutaneous test. o indicates a typically negative intracutaneous test. p.d. indicates that the animal died before satisfactory reading could be obtained. BIBLIOGRAPHY. 1. Nut t a l l and Graham-Smith, 1908, The B a c t e r i o l o g y of D i p h t h e r i a . 2. Neisser, 1913. 3aoteriologie der Diphtherie, Oentralbl. f. Bact., 57, Bhft. 1. 3. Kdlmer and Moshage, 1917. Methods of Determining Virulence of Diphtheria Bacilli. Jour. Inf. Die., XIX, 1. 4. Weaver, 1917. Virulence of Diphtheria Bacilli from Carriers and Patients. Jour. Inf. Dis. XX. 125. 5. Powell, Eoraoe M. 1923. A Biological Study of Diphtheria Bacilli. Am. Jour, of Hyg., III, 109. 6. Wade and Vaughan, 19 20. Observations on the Diphtheria Virulence Test in Public Health Work. Am. Jour. Pub. Health, X. 1426. 7. Foroe and Beattie, 1922. Intracutaneous Test for Virulence of Diphtheria Bacilli. Am. Jour. Hyg., Ill, 5490. 6. Weasbrook, Wilson and ScDaniel, 1699. Minnesota Public Health Reports. 9. Albert, Henry, 1920. A New Diphtheria Bacilli Stain. Am. Jour. Pub. Health, X, 334. 10. Bull and McKee, 19 23. Whole Culture Method of Testing the Virulence of Diphtheria Bacilli. Am. Jour. Hyg., Ill, 103. BIBLIOGRAPHY. (Continued). Havens and Powell, 1922. Use of Original Diagnostic Culture for Determination of Virulence of Diphtheria Bacilli. Am. Jour. Hyg., III, 234. Kelly and Potter, 1923. Administrative Value of the Virulence Test for Diphtheria Bacilli. Jour. Am. Med. Assn., LXXXI, 734. 

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