ANTIBODY RESPONSE TO HÆMOPHILUS INFLUENZÆ IN OBSTRUCTIVE AIRWAY DISEASE

1128 TABLE IV-PATIENTS FROM WHOM ISOLATIONS WERE OBTAINED:

RACE,

RESULTS OF FREI TEST, AND L.G.V.C.F.T.

In view of these facts and of the experimental evidence, it would appear justifiable to refer to the isolates of Bedsonia obtained in this study as isolates of TRIC agent. The results of the L.G.v.c.F.T. on the sera of the patients from whom isolations were made and the results of the Frei intradermal test in these cases are shown in table iv. Similar results, in these tests for immunological response to infection by the Bedsonia, have been obtained in studies of patients infected by TRic agent (Dunlop et al. 1964a and b). We consider that these results do not vitiate the conclusion that the Bedsonia isolated from the urethra in each of these cases is TRIG agent. The terms " inclusion urethritis " and " inclusion cervicitis " have not been used in this paper: they are unsuitable names because they imply that urethritis and cervicitis associated with infection by TRic agent are the genital counterparts of the inclusion conjunctivitis syndrome seen in adults, whereas it is known that some TRIC isolates of genital origin are capable of causing trachoma (Jones and Collier 1962, Jones 1964). They are also unsuitable because TRIG agent may be isolated from the urethra or from the cervix (Dunlop et al. 1964b), in cases in which inclusions have not been demonstrated. The terms " urethritis (or cervicitis) associated with infection " by TRIG agent " or, more briefly, TRIC agent urethritis and " TRic agent cervicitis " are therefore preferable. This pilot study shows that infection of the urethra by TRIG agent is associated with " non-specific " urethritis in some unselected patients presenting because of that condition. It indicates the necessity for a full-scale investigation of the role of this agent in all types of so-called non-specific genital infection.

Summary of " non-specific " urethritis in 9 men who presented because of that disease have been studied. Evidence of infection by TRic agent was found in 4. TRic agent was isolated in 2 out of 9 cases and inclusion bodies were found in urethral scrapings in 1 of these and in 2 more of the 10. 4 female sexual partners were examined. An inclusion was found in cervical scrapings in 1 case. Growth in yolk-sac, the results of electron microscopy, complement-fixation, and fluorescent-antibody tests showed that these two isolates, and a third obtained from a similar case, were members of the Bedsonia group. The pattern of growth in yolk-sac, the results of the inoculation of baboons and mice, and the absence of clinical evidence of past or present lymphogranuloma venereum in the 10 cases of urethritis, suggested that the isolates of Bedsonia obtained in this study were isolates of TRic agent. This view is supported by the fact that the clinical signs and the results of laboratory investigations of the disease, in the 4 cases in which there was evidence of infection of the urethra by TRIC agent, closely resembled those of the urethritis observed in the cases of the fathers of babies suffering from ophthalmia neonatorium due to TRic agent 10

cases

and in those of men presenting because of infection of the eye due to this agent. The terms " TRic agent urethritis " and " TRIC agent cervicitis " are proposed. We are grateful to Prof. C. F. Barwell, of The London Hospital, for kindly supplying the materials for the Frei test and for carrying out the L.G.V.C.F.T.; to Dr. A. E. Wilkinson, director of the V.D. Reference Laboratory of The London Hospital, for carrying out serological tests for syphilis and gonorrhoea, and cultures for bacteria, Trichomonas vaginalis and candida; to Mr. J. R. Kinnison and Mr. D. A. Knight for their valuable technical work; and to Mr. H. de C. Clarke for the electron micrographs. REFERENCES

Al-Hussaini, M. K., Jones, B. R., Dunlop, E. M. C. (1964a) Br. J. vener. Dis. 40, 25. (1964b) Meeting of the International Organization Against Trachoma, Vienna, 1964. See Rev. int. Trachome. (in the press.) Andrewes, C. H. (1964) Viruses of Vertebrates; p. 365. London. Bovarnick, M. R., Miller, J. C., Snyder, J. C. (1950) J. Bact. 59, 509. Dunlop, E. M. C., Jones, B. R., Al-Hussaini, M. K. (1964a) Br. J. vener. Dis. 40, 33. (1964b) Meeting of the International Organization Against Trachoma, Vienna, 1964. See Rev. int. Trachome. (in the press.) Findlay, G. M. (1933) Trans. R. Soc. trop. Med. Hyg. 27, 35. Gear, J. H. S., Gordon, F. B., Jones, B. R., Bell, S. D. Jr. (1963) Nature, Lond. 197, 26. Jones, B. R. (1964) Rev. int. Trachome. 41, 425. Al-Hussaini, M. K., Dunlop, E. M. C. (1964a) Br. J. vener. Dis. 40, 19. (1964b) Meeting of the International Organization Against Trachoma, Vienna, 1964. See Rev. int. Trachome. (in the press.) Garland, J. A., Dunlop, E. M. C. (1965) Unpublished. Collier, L. H. (1962) Ann. N. Y. Acad. Sci. 98, 212. Smith, C. H. (1959) Lancet, i, 902. Meyer, K. F. (1953) Ann. N. Y. Acad. Sci. 56, 545. Miyagawa, Y., Mitamura, T., Yaoi, H., Ishii, N., Okanishi, J. (1935) Jap. J. exp. Med. 13, 331. — — — — — (1936) ibid. 14, 197. Murray, E. S., Bell, S. D. Jr., Hanna, A. T., Nichols, R. L., Snyder, J. C. (1960) Am. J. trop. Med. Hyg. 9, 116. Thomason, B. M., Cherry, W. B., Davis, B. R., Pomales-Lebron, A. (1961) Bull. Wld Hlth Org. 25, 137. —

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ANTIBODY RESPONSE TO HÆMOPHILUS INFLUENZÆ IN OBSTRUCTIVE AIRWAY DISEASE

WILLIAM KEITH C. MORGAN M.D.

Maryland,

M.R.C.P.E.

WILLIAM H. WOOD M.D.

Sheff., Captain U.S.M.C. From the Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A., and the Department of Rickettsial Disease, Walter Reed Army Institute of Research, Walter Reed Medical Center, Washington, D.C., U.S.A.

evidence that the commonest type of emphysema-the centrilobular variety-is the result of repeated low-grade infections of the smaller airways (Leopold and Gough 1957). This insidious infective process is frequently referred to as chronic bronchitis and is notoriously prone to acute exacerbation which, in, patients in whom the disease is long established, is responsible for considerable morbidity and, occasionally, THERE is

some

death from cor pulmonale. In Great Britain there have been several bacteriological studies in which Diplococcus pneumonia and Haernophilus influenza have been isolated from the sputum of patients with chronic bronchitis (May 1953, Brumfitt et al. 1957, Murdoch et al. 1959), and after some initial difficulty these findings were amply confirmed in the U.S.A. (Brown et al. 1954, Davis et al. 1961, Norman et al. 1962). Antibiotics to suppress bacterial infection of the smaller airways have commonly been used in the treatment of chronic bronchitis with both symptomatic and clinical improvement; it is possible to clear up the purulence with antibiotics and to reduce the number of bacteria present

1129

in the sputum (May and Oswald 1956, Dowling et al. 1960, Norman et al. 1962). Nevertheless, while there is much circumstantial evidence incriminating H. infiuenzae as a pathogen, its exact role is not fully understood. For example H. influenze may be found in the sputum of patients who are in clinical remission; also, while the pneumococcus is suppressed fairly easily with most antibiotics, H. infiuenzae frequently persists, although in sparser numbers, despite the fact that both the volume and purulence of the sputum are reduced (Davis et al. 1961). We have used a different approach in that the part played by H. influenze in chronic obstructive airway disease was investigated with serological techniques. Methods

A complement-fixation test was devised by one of us (W. H. W.) using a soluble antigen of H. influenza prepared by the method of Tunevall (1953). For ease in examining large numbers of serum samples, the microtitre

technique was used. Preparation of H. influenzas Extract The strain of H. influenza used was A.T.C.C., type A-9006 (Margaret Pittman strain 610) and this was prepared by Tunevall’s method as modified by Glynn (1959). This strain was grown on Levinthal’s medium and collected after eight hours into 1 % sodium carbonate. After one hour at room temperature, the residue was removed by centrifugation and the supernatant fluid dialysed by running tap-water for twentyfour hours. The opalescent viscid solution was then centrifuged again and the supernatant fluid stored at 4°C.

Preparation of Standard Antibody were immunised with the soluble antigen of influenze (A.T.C.C., type A-9006) by a series of intravenous injections (1 ml. each) at weekly intervals. At the end of six weeks a 10 ml. sample of blood was tested by the complement-fixation test and, if a suitable titre had been obtained, the

Albino rabbits

H.

animals

were

bled and the

serum was

stored at -20°C.

Complement-fixation Test The complement-fixation tests were performed in transparent plastic plates using the microdroplet technique of Fulton and Dumbell (1949) and Takatsy (1950) as modified by Sever (1962).’Veronal’ buffered saline solution (pH 7-4) was the diluent. Standard 2 % suspensions of sheep-cells were prepared with colorimetric control. The haemolytic system consisted of equal volumes of a 2% suspension of sheep erythrocytes and a dilution of hxmolysin containing 3 units per 0-025 ml. (1 unitthe highest dilution causing complete haemolysis). Complement was titrated in the presence of antigen since the presence of complement alone might have influenced the amount necessary for complete haemolysis. Two full units of complement were used (1 unit = highest dilution of complement causing complete haemolysis, 1 full unit=next dilution of complement causing complete hxmolysis). The test sera were inactivated by heating to 56°C for thirty minutes and then serial twofold dilutions, beginning with undiluted serum, were made in veronal buffer. Antigen was titrated by the box method " and two units were used in the test (1 unit=maximum dilution of antigen giving 50% complement-fixation with the highest dilution of "

serum). In the testing of immune sera, the carried out as follows:

complement-fixation test

was

Serum dilutions (0-025 ml.) were placed in the plates. 0-025 ml. of antigen representing 2 units and 0-05 ml. of complement representing 2 full units were added from a pipette dropper to each dilution. After mixing the components, the plates were covered with ’Cellophane ’ tape and kept at 4°C for eighteen hours. The plates were then removed from the refrigerator, the tape was removed, and 0-05 ml. of the haemolytic system was added to each well. Each plate was thoroughly mixed and placed in a 37°C incubator for one hour. Included with each test were controls for positive and negative sera, antigen, hsemolysin, cells, and a titration for complement in the presence of

antigen.

The titres were expressed as the reciprocal of the highest dilution of serum giving a 50% fixation of complement. A titre of 1/4 or higher was regarded as positive; and, if multiple sera from the same subject were tested, positivity or negativity was based on the first specimen.

Technique of Isolation of H. influenzae In 13 selected subjects, all with asthma or chronic bronchitis, daily sputum cultures were made for six to eight weeks to allow correlation of the level of antibodies present with the presence of the organism in the sputum. This was done by drawing weekly blood specimens from these subjects whose sputum was being cultured. To compensate for the irregular distribution of organisms in the sputum all specimens were treated by Rawlins’ (1955) method. The bacteriological techniques are those described by Storey et al. (1964). Clinical Material Serum was drawn from a variety of patients-including a group with " chronic bronchitis " and patients with other types of chest disease-and a large group of controls. Reference to the first group as " chronic bronchitics " is not strictly accurate since sputum production varied widely among them. A few would even fall into the category of " dry emphysema ". For brevity we chose to refer to the whole group as bronchitics but this does not mean that we believe in a uniform aetiology for all cases of emphysema or that chronic bronchitis inevitably

accompanies emphysema. The controls

were

drawn from three main

sources.

The

largest control group consisted of sera drawn from a hospital population with various arthritic complaints and was made up of latex-fixation test negative sera which had been stored by the Division of Arthritis. The second group of control sera were drawn from pregnant women who attended the laboratory These sera for routine blood-grouping and rhesus-typing. differed somewhat in their serological response from the latex-negative group, and of course pregnant women are not comparable to chronic bronchitics so far as age and sex are concerned. For these reasons, although the results of the rhesustyped group are shown in the data, it was felt advisable to exclude them from the statistical analysis. The control group of patients with tuberculosis were also not ideal. As far as age and sex were concerned they were similar to the bronchitic patients; but their case-records showed that approximately 60% had symptoms of obstructive airway disease which in many instances was confirmed by lung-function tests. We also tested a large number of sera from asthmatic subjects whose mean age was less than that of the latex-negative controls and the chronic bronchitics; this group also included a greater proportion of females. Results

770 sera were tested, of which 105 were retested to see if the results were reproducible. Those sera with a positive titre on the first occasion remained positive on retesting with 2 exceptions. In 10 of the positive sera there was a difference in titre of one serial dilution on retesting, and in 4 there was a difference of more than one dilution. This was felt to indicate a satisfactory degree of reproducibility. All sera showing anticomplementary activity were discarded. This was present mainly in sera which had been frozen for over two years. The range of titres in the bronchitics and latex-negative controls is shown in table i. The higher titres were found in patients with obstructive airway disease, while those TABLE I-RANGE OF TITRES

1130 TABLE

II-770 SERUM SPECIMENS

respiratory-tract infections

TESTED

was found to have a positive several occasions. Her first positive titre occurred shortly after H. influenze began to appear in her sputum in large numbers. When the bronchitics were considered, those subjects who had a positive titre usually had H. infiuenzae in their sputum (see figure). In contrast,there was no obvious relationship between the number of H. infiuenzae colonies cultured from the sputum and the magnitude of the serological response; nor in those subjects who had

titre

control titres.

sera

which

were

positive tended

to

have lower

on

Apart from the controls, the sera tested came from patients with various pulmonary and other conditions. A breakdown of the total into their respective disease groups is shown in table

11.

When the results from individual patients in each group are considered, several interesting features become apparent (table ill). Patients with bronchitis have positive titres more often than do the other groups. The frequency of positive titres among the bronchitics is significantly higher than in the latex-negative controls (X2=2429;

P <0-001). The bronchitics, asthmatics, and latex-negative control groups are not strictly comparable so far as age and sex are concerned and the essential differences between the three groups are shown in table IV. Of the positive sera in the latex-negative control group, rather surprisingly only 12 out of 28 came from men. This suggests that the greater proportion of females in the control group did not influence the results. Moreover, if males only are considered (table v), the results are still significant (P < 0-001). TABLE III-DIAGNOSTIC ORIGIN OF POSITIVE SERA FROM INDIVIDUAL

PATIENTS

In the asthmatic patients there was no correlation between the serological response and the presence of H. infiuenzae in the patient’s sputum. This is illustrated by reference to 1 patient who in a seven-week period had only one positive titre; yet during the same period H. influenzce was isolated from her sputum on at least three occasions each week (see figure). At one time this organism was isolated on seven consecutive days and yet her complement-fixation was still negative at the end of this period. In general this relationship held true, however; one asthmatic subject who produced more sputum than the other asthmatics and who was also prone to lowerTABLE IV-SEX AND AGE CHARACTERISTICS OF THE THREE MAIN GROUPS

Correlation of positive complement-fixation of H. inftuenzae in sputum.

The shaded area indicates the number of H. influenza was cultured.

(C.F.T.) with

days in

presence

each week when

antibodies present in their serum was there an obvious rise in titre at, or shortly after, the onset of an acute exacerbation. Nevertheless, as the sputum became more

purulent, there was often a change from a negative to a positive titre. Those subjects who produced the greatest volume of sputum were most likely to have a positive titre, and conversely, those subjects who had what is commonly referred to as " dry emphysema ", where sputum production was a minor symptom, were found less likely to have a serological response. No correlation was evident between the level of antibody titre and standard tests of ventilatory function. Serum was obtained from several patients with acute pneumonia, or convalescing from pneumonia. In 3 of these patients, H. influenzae was isolated from the sputum during the acute illness although there was no reason to believe that this organism was responsible. In 1 patient a weakly positive titre (1/4) was found in the serum during convalescence. This patient also had chronic obstructive airway disease. 3 out of 5 patients with bronchiectasis had a positive titre. Steroids did not influence the antibody response in the asthmatic subjects but it is more difficult to be certain of their effect in the bronchitic subjects as only 5 of the latter received these drugs. One bronchitic with a persistent positive titre received steroids for four weeks with no apparent effect. His complement-fixation test was positive before, during, and after the medication; the titre varied between 1/8 and 1/16. Antibiotics did not influence the antibody response. Discussion

TABLE V-TITRES

AMONG MALE BRONCHITICS AND MALE

CONTROLS

In the past, H. influenzae infections such as meningitis have been investigated by serological techniques. These have made use of type-specific antigens and transient specific agglutinins to autogenous strain of H. !M/?M6M< have been demonstrated. We tested all sera against an extract of H. influenza prepared by the method of

1131

Tunevall (1953), who claimed that it contained speciesall strains of this organism. specific antigens of in chronic obstructive airway studies The only report is of that disease Glynn (1959), who used the tanned-redHe demonstrated a difference between cell technique. people with chronic bronchitis and controls, but did not show a rise in titre following exacerbations of bronchitis. Much of the research in obstructive airway disease has been restricted to the epidemiological features and the respiratory impairment found in this condition. A fresh approach seems to be desirable. Recently, it has been suggested that subjects with irreversible obstructive airway disease fall into two clinical types-" the pink puffer " and " the blue bloater ", the former being the patient with dry emphysema while the latter is the chronic bronchitic (Dornhorst 1955). It seems that serological studies have an obvious application in the investigation and characterisation of the different types of obstructive common to

airway disease. Although there is a wealth of circumstantial evidence to incriminate H. influenzce in exacerbations of chronic bronchitis, there remains a sceptical minority who are not convinced that the organism is pathogenic or indeed that infection plays much of a part in chronic irreversible airway disease (Davis et al. 1961, Wright and Kleinerman 1963). While Davis et al. (1961) accept that H. influenza is to be found in the lower respiratory tract of most subjects with obstructive airway disease, they feel it is present as a commensal only and has no more significance than does the presence of Neisseria catarrhalis. On the other hand, even among those who accept the fact that H. infiuenze is partly responsible for the acute exacerbations of chronic bronchitis, doubt exists as to its exact role. Is primary bacterial infection with D. pneumoniae and H. influenzee the sole cause or do other factors playa part ? Apparently many of the exacerbations are brought on by previous viral infections, which probably damage the bronchial epithelium of the lower respiratory tract and allow secondary bacterial infection to occur (Carilli et al. 1964). Air pollution could perhaps act in a similar fashion. Nevertheless, whatever the precipitating factors, there is little doubt that bacterial infection, whether primary or secondary, does play a role in chronic bronchitis, and our results add further weight to this view. Moreover, it seems that H. influenze may be present in the lower respiratory tract of subjects with obstructive airway disease without obvious harmful effects and without engendering an antibody response. It also seems that, in those bronchitic patients who have a positive titre, the organism is no longer acting as a commensal but has assumed invasive properties. A demonstration of a rise in titre with exacerbation of the disease would support this suggestion. A more detailed study of the properties of the antigen is also required.

Summary

Serological techniques with an antigen prepared by the (1953) have been used to study in Haemophilus infiuenzae airway disease. Serum from patients with chronic bronchitis commonly contains a species-specific antibody to H. influenzae. This method of Tunevall

response is far less usual in control

serum.

Serum from asthmatic subjects resembles the control group in that it has less often a positive titre. In the asthmatic group a negative titre in the blood was common despite the concomitant isolation of H. influenzz from the sputum.

The presence of bronchitis as manifested by production of a purulent sputum, seems to be related to the presence of antibodies to H. influenzce in the serum. Bronchitics who were serologically negative often developed a positive titre after an increase in sputum production, but acute exacerbations of the disease did not cause a rise in titre. Pneumonia and miscellaneous pulmonary conditions were not associated with a positive antibody response. We thank Dr. A. Schubart, head of the Division of Arthritis, for making available the latex-negative control sera; Mr. J. Hatgi and Mr. B. Harrington, who carried out most of the complement-fixation tests; and Dr. W. Reinke for statistical assistance. This work was supported by grant AP0045 from the Division of Air Pollution, Bureau of State Services, U.S. Public Health Service, and by a grant from the Maryland Tuberculosis Association. REFERENCES

Brown, C. C., Coleman, M. B., Alley, R. D., Stranahan, A., Stuart-Harris, C. H. (1954) Am. J. Med. 17, 478. Brumfitt, W., Willoughby, M. L. N., Bromley, L. L. (1957) Lancet, ii, 1306. Carilli, A. D., Gohd, R. S., Gordon, W. (1964) New Engl. J. Med. 270, 123. Davis, Anne L., Grobow, Evelyn J., Tompsett, R., McClement, J. H. (1961) Am. J. Med. 31, 365. Dornhorst, A. C. (1955) Lancet, i, 1185. Dowling, H. F., Melody, M., Lepper, M. H., Jackson, G. G. (1960) Am. Rev. resp. Dis. 81, 329. Fulton, F., Dumbell, K. R. (1949) J. gen. Microbiol. 3, 97. Glynn, A. A. (1959) Br. med. J. ii, 911. Leopold, J. G., Gough, J. (1957) Thorax, 12, 219. May, J. R. (1953) Lancet, ii, 534. Oswald, N. C. (1956) ibid. ii, 814. Murdoch, J. M., Leike, W. J. H., Downie, J., Swain, R. H. A., Gould, J. C. (1959) Br. med. J. ii, 1277. Norman, P. S., Hook, E. W., Petersdorf, R. G., Cluff, L. E., Godfrey, M. P., Levy, A. H. (1962) J. Am. med. Ass. 179, 833. Rawlins, G. A. (1955) J. clin. Path. 8, 114. Sever, J. L. (1962) J. Immunol. 88, 320. Storey, P. B., Morgan, W. K. C., Diaz, A. J., Klaff, J. F., Spicer, W. S. (1964) Am. Rev. resp. Dis. 90, 730. Takatsy, G. (1950) Orvostud bes. 2, 393. Tunevall, G. (1953) Acta path. microbiol. scand. 32, 258. Wright, G. W., Kleinerman, J. (1963) Am. Rev. resp. Dis. 88, 605. -

COLOUR-BLINDNESS AND CIRRHOSIS OF THE LIVER R. CRUZ-COKE M.D. Chile OF THE DIVISION OF

MEDICINE, HOSPITAL J. J. AGUIRRE, UNIVERSITY OF CHILE, SANTIAGO

THE genetic basis of a common disease may be studied by considering its prevalence in two groups distinguished by the presence or absence of some gene of high specificity (Edwards 1963). The association of genes at autosomal loci, such as those of blood-groups ABO, Rh, MN, with

diseases such as duodenal ulcer, cancer of the stomach, or rheumatic disorders, have been investigated (Reed 1961). The strongest association of this kind yet discovered has a relative prevalence of only 1-4. This genetic " or predisposing risk is small, considering that the relative risk of lung cancer with cigarette smoking is between 9 and 10; that is, the risk of lung cancer is 800-900% greater among smokers than non-smokers (Lilienfeld 1961). Nevertheless, the discovery of these associations may throw light on the cause of the diseases involved (Clarke 1962). The present study revealed a strong association between a sex-linked gene and an important common common

"

disease. Patients and Methods The sample consisted of 450 male and 450 female patients who were admitted to this hospital between Oct. 1, 1963, and Sept. 1, 1964. Only severely ill and blind patients were excluded. The age distribution of the sample is similar to the age distribution of the total yearly hospital population. During their first week in hospital, all these patients were