od IRECTOR OF THE HOSPIT FT 0 INSTITUTE FOR MEDI EARCH January 1919 To the Board of Scientific Directors of The Rockefeller Institute for Medical Research. Gentlemen: During the past quarter the same conditions that have restricted the research activities of the Hospital during the past year have continued to be operative. In addition to the absence of many of the staff, Dr. Van Slyke has been 421, and consequently there has been little work accomplished in the chemical laboratory, aside from routine work and teaching. Dr. Cohn ie still abroad so that the work in his department has been practically at a standstill. The interne staff has consisted entirely of amy officers detailed to the Hospital. These men are on temporary duty, subject at any time to orders sending them elsewhere. Most of them have had little or no training in research methods. Consequently it has been practically impossible for them to .. engage, in investigation. . On account of the character of the. interne Riba staff, it has been necessary for the Director. to. give much of: his time -to the actual diagnosis and treatment of, patients in the wards..:.-: eters ee ... the work of training members of the army medical. corps has continued during the quarter and has occupied a considerable amount of the time of the staff. This work, as mentioned in the previous reports, has consisted of four especial courses: aes \ I,-A course in bacteriological methods given for medical offi- Cers in the Institute Laboratories. The instruction of the men in the methods of studying the bacteriology of acute infections has been given by Dr. Avery and Dr. Doches, assisted by Dr. Stillman, and this has required a considerable amount of their time. II.-A course in chemical methods for members of the Sanitary Corps has been given in the chemical laboratory of the Hospital. III.-A course designed to teach women the bacteriological methode employed in the etiologic diagnosis of acute respiratory diseases has been given during the past quarter in the Hospital. This course has been under Dr. Avery and Dr. Stillman. IV.-Training medical officers in clinical methods. These offi- Ceres have also acted as internes. All these courses have now ended and it is not likely that there will be any further need for this kind of activity at present. Gaty elacwhere. na : . 7 . S cy eben : Bat has 7 he Bt tbe eho gy , oa - va hoa ee ee, Dy af Ps ska eater? Lee “any REE WONG Oo wey ag ae uefa Ss wa org Abhesliow Fee chts In: spite: of the, conditions during the war, ;it. has; been. possible to maintain the essential: character of the Hospital, and,with. . ' the. coming: of peace, it.seems likely that:a permanent. staff can soon ber ots oO assembled and that:the work of the Hospital can proceed in orderly fashion once more. Nevertheless, much reorganisation will be necessary, and it seems advisable not to attempt to obtain a full staff at once, nor to attempt: to resume all the lines of work in progress at the opening of the war. Se much depends upon obtaining exactly the right kind of men to assist in the - work that it seems'advisable to resume the work and to replace the staff gradually as the problems develop. +t Acut i ry Di . The previous report was written very soon after the epidemic of influenza had made its &ppearance in the army campe and when it was beginning in New York. Since then, this disease has pre- vailed to a very great extent in New York. We have, therefore, been com- pelled by circumstances to extend our study of acute lobar pneumonia to the study of influensa. Indeed, the character of the acute pulmonary infec- tions in New York has #0 changed during the past year that now typical lobar pneumonia in our experience has been largely replaced by pulmonary infections complicating influenza and by atypical inflammations of the lung. \ We have admitted to the Hospital a considerable number of persons suffering from influensa in whom no extensive pulmonary lesions have developed. These Were largely members of the staff and work- ing force of the Institute. These patients offered excellent material for study of the ‘milder uncomplicated cases of influensa. . Ae far as possible, , however, ‘the ‘gimtesion © of patients: ‘trom “the. outside was Gonfined to ‘those 2 BS Veta RO Gti ned oe hemhad aF ali Rouen cates Oba in whom: pulmonary lesions were present or were suspected at the’ ‘tine’ of Meta To be eawtaly thet Anfivtnee heaoih ts Pe gol peine ovaelee “ edaieaton. ‘Yor this reason, the disease as. seen in the Hospital. was’ of ak Pita TAL aoe 20 many cars ws mae rey somewhat more severe character than the ‘Qisease’s as ait “existed in the eity at large. “this is’ ‘evident ‘trem the ‘high’ percentage of phouonia ‘and’ high Be fof . “ a ad wat Hye wa mortality of the’ disease : in the Hospital. oe ae observation of the earliest cases made it evident that the etlology of the disease was “uncertain, With the depleted staff and other work demanding attention’ it was obvious that “at was impos- sible for us ‘to: investigate the etiology from all standpoints. It there- fore seemed advisable for the Hospital staff to confine its attention ~ mainly to the study of the réle of the influensa bacillus in the epidemic and to the investigation of the other microorganisms concerned in the pul- monary complications, leaving to members of the Laboratory Staff the search for an unknown virus. Dr. Amoes and Dr. Oliteky, therefore, under- took the latter problem, obtaining material for this investigation from the Cases treated in the Hospital. Their results are given in the report of the Director of the Laboratories. The attempt has been made in the Hospital to study with as great care as possible the predominating organisms present in the throats and sputum of all the cases of influenza and pneumonia during life, and to study the bacteria present in the lungs at autopsy. It has been the intention to make this study with as great accurucy as possible on the limited number of patients coming under our observation. Thies has involved a larger amount of work than might seem necessary at first sight. There were, at first, many technical difficulties, eapec- ially in the isolation of B. influensae. We have thought it necessary in all cases, in order that we might be certain that we were really dealing with this organisn, that it be obtained in every Case in pure mlture and that ‘the organien 80 obtained be tested by all known nethods, Moreover, — in order to ‘be certain that : “Anfluensa bacilli Were not being overlooked, it was, ‘necessary. in amy tas cases %, ‘make repeated cultures. With greater experience and especially y with th the improvement. 4: in’ method of isolation de-~ vised by Dr. Avery, ‘of which ‘mention will be. made. later, the difficulties . . in the study have been much diminished. ° : : | The following table shows the frequency of occur- / rence of influensa bacilli and other predominating organisms in the cases of influensa and paetmonia, studied in the Hospital during the past quarter. Uncomplicated influenza | Total number 49 a a “sy : eae t et is 4 he B. influsncae present 4 Other Predominating Orgeni ems Pneymococct Staphylo- I {II /Imj;IV 2); 2 |17 coccd Norm-kemolytic streptococe! Hemolytic streptococct lander Died —— Z Influenza with pneumonia 43 27 12 28 a Bs faa wet[o Bs e Atypical pnev- . Oe monia me 8 Fel 1| 4 2 1 2 33 influenzae (7) : * fe Lobar pnew- = ie ‘s * - - monia 382 [= 55 3/ 2); 8/2]; — = — — | 2 | 3 & Yo © OO Pee leg |? : 4| 4/12 | 65 6 3 2 1 21 18% ok 2S Bop eS Ss ao BS Tee ee race mo ecg arcane ates ne ean en ee naman nl eat cr In most cases cultures were made from the throat and sputum, and in the cases coming to autopsy, from the various organs. The cultures were made by various methods, by mouse inoculation, culture on blood agar plates and on oleate agar. The latter method proved by far the most successful. The most striking fact brought out is the fre- quent occurrence of B. influensae in the influensa and broncho-pneumonia cases. In the cases of lobar pneumonia, B. influensae has been encoun- tered less often than in the influensa cases, and with more nearly the sane frequency as in normal throats during the same period. Hemolytic strepto- cocci have been found very seldom, in only two throats. An interesting fact is that in six cases staphylococci were present in large numbers, in five of the pneumonia cases they werd the predominating organism and seemed to be responsible for the complication. The clinical features of the cases have not yet been carefully analysed. No striking clinical differences have been present in the Cases ot Ant luensa here a those described in a cases occurring | elsewhere, ; Yo extensive study ot the blood changes have been undertaken. Fairly frequent blood counts have deen mate in a routine manner on all cases, Most of the cases have shown the leucopenia which has been ‘generally observed, Distribution 5. Bs influenzae in the Mouths af Normal, Persons _ and_of Convalescents. eo a : PoP A LE Mak 2 On account of the great prevalence of B. inf luen- eet a sae in the cases of. pneumonia and inf luensa oceurring in the ‘Hospital it nes aie tt seemed of importance to determine the frequency of their « occurrence in} nor- mal mouths during the same period. It was planned to make a complete sur- vey of the mouth flora of the workers and staff of the Institute, and this | work has been undertaken by Dr. Stillman, assisted by Miss Pritchett. Sone time was lost in developing a proper technique for isolating B. influenzae but the work is now progressing at a fairly rapid rate. Unfortunately Mise Pritchett must leave to undertake her new duties at Baltimore but her , work will be continued by Miss Winchell, and it is hoped that by making fairly frequent cultures, curves can be made of the occurrence of carriers of B. influensae in the various departments during the coming months. It is realised that this Institute is not ideal for such a survey ae the popu- lation 1s somewhat shifting, and since most of the persons working here have wide contacts outside the institution. Nevertheless, the advantages of making such a study where the facilities for work are #0 satisfactory, and where the residents can be constantly under the observation of the investi- gators, seem to outweigh the disadvantages, and it is hoped that such a eur- vey may throw some light on the distribution of influensa and corysa, and possibly on the etiology of the disease. Up to the present time, a study has been made of 231 individuals. In most instances cultures have been made only once, and the present figures indicate roughly the conditions present in this Insti-— tute during the latter part of November ane Recenter a 7 of the 231 persons, 54 had suffered from influensa during the ee three monthe and should therefore be considered convales~ ‘cents, 177 gave no history ot having had this disease. From the sputum or . throate of 42 ‘per cent of these normal individuals, influenza bacilli have been isolated. of she 54 persons in whom cultures were made during conval- escence from influenia, 46 per cent showed the ‘presence of B, influensae in the throat. | . Almost one half the persons in the Institute who have been studied during the past months, therefore, have carried B. influ- ensae. Whether this wide distribution is constant or is only associated with the presence of the epidemic, only further studies will show. The latest studies indicate, however, that the incidence of this organiem in normal throats at present is much less than it was a couple of months ago. Moreover, the distribution of the carriers of this organism has been inter- esting. For instance, in the preparation room, where there were two con- valescent carriers, all of the eight persons working there carried this organism. On the other hand, in the chemical laboratory, none of the workers were found to harbor B. influenzae. Strangely enough, very few carriers have been found among the nurses in the Hospital. It is obvious that no conclusions should be drawn from this study. at the present time. The line of investigation, however, seems promising. Study of Biological Properties of B, influenzae. Dr. Doches and Dr. Avery have been engaged in a study of the properties of the influensa bacilli isolated in this Hospital, One fact observed which is of much practical importance is that the growth... of this organism is.enhanced by the addition to media of certain soaps of _ the unsaturated: fatty acids... This favoring action to growth seems exerted | generally in the case of Gram negative organisms and the opposite effect is os observed in the case of Gram. positive organisns,: especially streptococcus of. 4 and pneumococcus,:: Use, has deen made. of this. observation in the production. of @ selective: wedium for B. influensae. ...The medium. is prepared bythe saws addition of, sodium oleate to 2 per.cent meat infusion agar in such amount as to make the concentration of oleate 1 to 1000. The initial hydrogen. ion concentration of the agar should represent a pH of 7.3 to 7.5. To 100 parts of the agar, while hot, there is added 1 cc. of a suspension of rabbit's corpuscles. Because of the more luxuriant growth upon this medium, the . eZ oe XK ordinary blood agar, and in later stages of development are not infrequent- colonies of B. influensae appear larger and less translucent than upon ly nucleated. Upon this medium, the Gram negative cocci of the catarrh- alis group, staphylococci, and occasionally diphtheroid bacilli, grow, while pneumococci and streptococci of the hemolytic and viridans group fail to develop. The use of this medium has very greatly facilitated the atudy of the eccurrence and distribution of B. influensae. A further observa- tion has been made that Gentian violet in dilution of 1:100,000 - 21:50,000 still further restrains the growth of Gras positive organisms, eliminating staphylococcus, without materially influencing the growth of B. influensae. Tt has also been noted that, contrary to the general belief, the influensa bacillus lives for as long a period as eix weeks in blood broth without transfer in the incubator at 37° C., at room temperature, and at 8° c. | It has deen found possible to raise the virulence of B, influensae by passage through rate and mice, so that 1/50 co. of a 24 hour blood broth cultures will kill a rat and .001 cc. prove fatal for white mice. Under these conditions the organisms are found in great numbers in the hearts’ blood of these animals... . ce dale tet Poeeh Sacaie suns? @ vpiosiiy with Type 11 Remmentation Reactions. 2 ncn wale nf tens Avery, . Preliminary, study of the fermentative reactions of © B,, influensae, indicates, that, this organiaa forms little .or ;n0 .a0 1d :fromvar~ 4 ious test substances, used. .;, Because of this. tact use, cannot be made of car- : vohydrate media. containing the,usual.acid indicators... Use.is therefore:sra being made of the method for determining the hydrogen. ion concentration of wt cultures of this organism. . ... ce Soe A, - Toxin Production. A study has been undertaken to determine whether or not B. influensae by growth in fluid media produces a soluble toxin of wi _~ any considerable degree of potency. Such studies up to the present have in the main been negative. It has, however, been possible to produce in fluid media a substance of sufficient toxicity to kill white rats in doses of 0.5 cc. of the bacteria-free fluid. In the course of bacteriologic study of patients in the wards suffering from influenza, it was found that in addition to the typical B. influensae, a different Gram negative hemoglobinophilic bacillus was present. Thies differe essentially from the influenza bacillus in the following characters: greater uniformity in staining property, marked alkalin fermentation of milk with subsequent peptonisation and active hemolysis of blood celle. A further study of thie organism is being made. Dr. Lyon has undertaken the artificial production of immunity in rabbits to virulent cultures of B. influenzae. This work has not yet progressed far enough for a report of the results to be made. Further Study of Atypical Type II Pneumococci. It has bem found that a considerable number of pneumococci isolated from cases of pneumonia, and also from normal mouths, react atypically with Type It serum, ‘Some ‘study has been made of these organiams by Dr. Avery, the results of which have been previously reported, and which have been published. From a study of ten of these strains Dr. *) Avery was able to classify them into three types, II a, Il b, and II x. By means of agglutination, absorption and protection experiments, the nenbers- .of sub-types II a and II b were found to possess immunity reactions identical with all other strains of the homologous types. Sub-type II x, however, consists of a heterogeneous series of independent strains which showed neither cross-agglutination nor cross-protection. Dr, Stillman has collected 204 strains of these reg atypical type II pneumococci and has made a complete study with the purpose of elaborating further their Classification on the basis of their specific relationships. The source of the strains studied was as follows: Lobar pneumonia 77 Post-operative pneumonia 2 Meningitis 1 Guinea pig pneumonia 5 Normal mouths 100 Convalescent Type I pneumonia 6 n ® II " 3 " * Ill 7 1 Dust —h. | Total 204 In classifying these strains agglutination and absorption reactions have been employed. On the basis of specific agglu- | ination in monovalent rabbit serun, the 204 strains have been classified into twelve distinot groups. Only two strains showed cross-agglutination _in the. immune serum of.a heterologous. group, . and: these reactions were shown to be due to the- presence of minor. agglutinins. for one group. og | ee ty “on... i, Organiems belonging to certain sub-groups, namely, . Il @ and II h, have been encountered. much more frequently. in relation to . - disease and it is probable that these have a greater pathogenicity. Organ- iems of these groups occur rarely in normal mouths.. Just the Opposite te the case with organisms of ‘certain other groups such as groups II b, IIc, II f, and II m. Organisms belonging in these groups are much more frequent-: ly encountered in normal mouths than in association with disease. on NN Biologic Classification of Streptococcus Hemolyticus. In. my previous report mention was made of the study being carried out by Dr. Dochesz and Dr. Avery on hemolytic streptococci, and it was then stated that evidence had been obtained that the various strains of haemolytic streptococci differ in their immunologic reactions, and that these organisms may be grouped on the basis of common characters, just as is the case with pneumococci and meningococci. While the relations between the various types of streptococci are more complex than is the case with pneu- mococeci, and while there have been considerable technical difficulties to be overcome in working with streptococci due to (1) the difficulty in rendering them virulent for mice, (2) their tendency to spontaneous agglutination, (3) difficulty in immunising animale, nevertheless, the work has progressed favor- ably, a considerable number of strains have now been thoroughly studied, and the results of the investigation will sooa be ready for publication. t wi 9 ‘Drs Stillman The quarter Szenes on Ooteber 1, 1918, with fatty. | Fetes ce ela yon U8 four ayphinitic ‘patients ‘in the Hospital. aun of then with two exceptions iw 5 were soldiers or enilore. Since Oatober a, thirteen patients have been 1 7 cee herey adnitted to the Hospital, ‘the nunber ot adniesions being curtailed on account Grae stak B Toot Be, ‘ of the recent epidemic of influensa and ‘the need of hospital room a these cases. Fifty-three patients have been discharged from the Hospital during this period, 80 that seventeen syphilitic patients were in | the Hospital on the first of January 1919. Of the fifty-three patients dis- charged, forty-five left as improved, that is with a negative Wassermann ae a ~ \ reaction both in the blood and in the spinal fluid. Four were admitted to the Hospital with a diagnosis of syphilis but were found to be not syphilitic. Two were transferred to other hospitale on account of complicating infections, and the only eyphilitic patient who was discharged with a positive Vassermann was a congenital syphilitic who is to return later for further treatment. During the month of October there occurred three severe cases of dermatosia directly attributable to treatment with A 189. Toward the end of October, 1918, one of these cases died of acute exfoliative dermatitie. There also occurred two cases of severe acute conjunctivits. Tne occurrence of these complications made it seem incumbent to revise some- what the mode of application of the drug, so that since October 20th the drug haa been given at two week intervals in doses of seven milligrams per kilogram of body weight. It has been dissolved in 2} molecules of sodium hydrate and 750 parts of saline solution. The analysis of results presented in the October report indicated that variations in alkalinity and dilution within the limits used have no effector the reactions occurring in the administration of A 189; and therefore ‘the “fact that ‘during ‘this quarter ‘we ‘have ‘had nd severe reactions may ‘be attributed 'to’a ‘lengthened ‘interval ‘between doses.'"* The ‘lengthened in- terval between doses may be accountable ‘also for the two following conditions which have ecourréd- “liter recent ‘treatmentras 6 toeoy of tha tipestton af . 1°}), “vIn two cnges ‘mucous ‘patches ‘have ‘recurred ‘directly ‘under ‘our’ observation, from:each of which ‘Troponemate pallida have been isolated.~* i. .~ 2,..Two cases have developed béth clinical and laboratory findings . of central nervous syphilis under treatment; but were definitely known not. to” have either before the institution of treatment. ' It appears possible, therefore, that the diminution cee , You os \ in toxic effects, gained by administering A 189 at approximately half the previous rate, may be attained at the cost of lowered therapeutic efficiency. Chemical Laboratory. Owing to Dr. Van Slyke's illness and the reduced staff, the research activities in the chemical laboratory have been markedly diminished during the past quarter. Dr. Cullen has been continuously em- ployed at the War Demonstration Hospital. It is hoped that he will soon be free to resume his work here. A considerable amount of the space and facil- ities of the chemical laboratories have been used during the past quarter by the men of the Sanitary Corps taking the course in clinical laboratory methods under the direction of Capt. Robinson. Mise Hiller has continued the work on which she was engaged with Dr. Van Slyke when we was compelled to leave. This problem consisted in ascertaining the optimum nitrogenous nutrient for pneumococci. Neither intact proteins nor the amino acids resulting from their complete hydrolysis appear to be as good for pneumococcus media as some of the prod- — ucts of partial hydrolysis, termed, indefinitely "peptones™. _ the object of Miss Hiller's work is to ascertain how completely it is desirable to digest. | the proteim pabulum in order to get it into optimum condition for the bacter- ia to grow on. The preliminary work has been a study of the digestion of casein with trypsin under accurately reproducible conditions, the curve of digestion being followed by amino nitrogen determinations, so that it is pos- sible to produce at will a digest in which hydrolysis has reached 10, 20, 30, or 40 per cent of completion to the amino acid stage. The digestion is stopped at the desired stage by addition of acid, and after autoclaving the solution is filtered and the hydrogen ion concentration brought to the point desired for pneumococcus cultures. Dr. Avery is about to test media in which peptones of varying degrees of hydrolysis, prepared as above described, are used as nitrogenous foods for the bacteria. Dr. Stadie and Capt. Rotinson have been studying the blood gases in patients with influenza and pneumonia, the oxygen and carbon dioxid in not only the venous, but algo the arterial blood being determined. The methods for the determinations are those recently devised by Dr. Van Slyke. Normally the arterial blood seems to be completely, or almost completely, saturated with oxygen, although the number of analyses of normal blood is not sufficiently great to make this certain. Influenza patients with cyanosis frequently show as low as 75 per cent of their arterial hemoglobin saturated with oxygen, and in one fatal case, only 25 per cent, The loss of oxygen which the blood suffers in passing from arteries to veins 1a in some cases also greater than usual, indicating the probability of a slow circulation, which in such cases would also be a factor in producing the cyan- ovis. The above conditions are indicated rather than proven, for the work is in ites preliminary stage; but it appears to promise results of both path- ological and physiological interest. The work is a logical outgrowth of ‘that done in the Hospital a year ago by Lundsgaard on the oxygen unsaturation of the venous blood in normal persons and cardiac patients.