THE DISTRIBUTION OF TYPHOID Vi AGGLUTININS IN NORMAL SERA WITH SPECIAL REFERENCE TO THEIR DIAGNOSTIC VALUE

IN TYPHOID FEVER

By D. W. SOMAN

{From the Haffkine Institute, Bombay)

Isolation of the causative organism from

blood, urine or feces of a patient, remains even to this day an unquestionable evidence of disease such as typhoid fever. However, when that is not practicable, reliance has to be placed on the serological diagnosis using the conven- tional Widal test, the correct interpretation of the results of which is beset with many difficul- ties. The discovery by Felix and Pitt (1934) of a Vi antigen of B. typhosus, and its serum-

counterpart Vi antibody in the sera of typhoid patients and carriers, opened up a new serological

approach to the problem of diagnosis of typhoid infection. Bensted (1937) and many other workers since then have confirmed the findings of Felix and his co-workers and acknowledged the importance of Vi agglutinins in the diag- nosis of typhoid carriers; but the demonstration of these agglutinins involved a laborious

technique of preliminary absorption of H and 0 agglutinins, which made it hardly a practical proposition to be used in the routine work.

Bhatnagar, Speechly and Singh (1938) overcame these difficulties by finding out a pure non-motile strain of B. typhosus Vi I Bhatnagar, rich in Vi antigen and possessing only a trace of 0 antigen. Using this strain, Bhatnagar (1938) showed the development of Vi agglutinins in acute typhoid cases, whether uninoculated or inoculated, to be very regular, provided the tests were done at

proper intervals, during the course of disease. But such repeated examinations of blood, though possible among men under military discipline and in hospitals, could not be done in private practice, and reliance had to be placed on a

single serological examination. Under these

circumstances, of what value Vi agglutination test could be was not known, and since this Institute was giving free diagnostic aid to

medical practitioners, in the diagnosis of typhoid infection, it was decided to take this opportunity to examine such sera for the presence of Vi

agglutinins. An accurate knowledge of the limits of natural Vi agglutinins among the local population is essential to the determination of the lowest significant Vi titre, and in view of the paucity of published data on the subject, it was considered desirable to secure information

concerning the distribution of Vi agglutinins in normal sera in Bombay population. Three hundred random samples of blood

received for Wassermann reaction were tested for the presence of Vi agglutinins. Only clear sera, free from htemoglobin, were used for the tests. The antigen used was a live suspension of B. typhosus strain Vi I Bhatnagar, prepared from a 24-hour culture on saccharose-agar, and the strain was maintained according to the instructions given by Bhatnagar in his paper. The suspension had an opacity of about 8,000 millions of organisms per c.cm. In addition to

Feb., 1945] DISTRIBUTION OF TYPHOID Vi AGGLUTININS : SOMAN 91

this, a preserved agglutinable Yi I suspension from the Enteric Laboratory, Kasauli, was used in parallel, in the earlier part of the investiga- tions. The suspensions, both living and killed, Were required to satisfy the criteria of sensitivity to Vi sera in high dilutions and non-agglutin- ability in pure O and H antisera, even in a

dilution of 1 in 5. Dilutions were put up in Felix tubes and ranged from 1 in 5 to 1 in 25 in case of normal sera. Dreyer's drop technique was used throughout, and one drop of the suspension was added to each tube of serum-saline mixture, which amounted to one c.cm. The racks were Well shaken and incubated at 37?C. for two hours and were left at room temperature over- night. Readings were taken next morning according to the instructions given by Bhatnagar. Each tube was examined naked eye as well as with a magnifying lens, and the reading was confirmed by gently shaking each tube. Three hundred samples of normal sera were

thus examined for the presence of typhoid Vi agglutinins. The results are set forth in table I.

Table I

Vi agglutination in 300 normal sera

Agglutination ( Number ot Pcicentag litre'against. sera | agglutination strain Yi I

1 ir* 5 to 1 in 25

1 in 5 17 5.65 1 in 10 4 1.35 1 in 16.5 1 0.3 1 in 20 0 0.0 1 in 25 3 1.0

8.3

.The information concerning the distribution of *i agglutinins in normal sera is very scanty, and among the few workers who have published the rePorts are Pijper and Crocker (1937), Lewin 1938), Horgan and Drysdale (1940), Davis (1940), Radowsky (1942) in different parts of ?frica; Eliot (1940) and Coleman (1942) in

y-S.A.; Bhatnagar and his co-workers (1937) in lndia. The number of normal sera showing Vi agglutinins varied from 2 to 10 per cent, in the hands of different workers, in the absence of any uniformity of technique; the technique varied essentially in regard to the range of dilutions put

the strain of B. typhosus used, for the pre- paration of suspensions. In this investigation, as pointed out before, a Vi I strain of B. typhosus Was used throughout, and the technique followed essentially the instructions given by Bhatnagar Personally. The results obtained from table I early indicated that only in 25 out of 300

jj?rmal sera (8.3 per cent), Vi agglutinins could 6 demonstrated. Seventeen out of 25 such sera ^ere positive in a dilution of 1 in 5 only. It was |^t possible to obtain any samples of faeces from he Vi positive persons; therefore the significance ?* the positive findings could not be further

estimated. With 92 per cent of normal sera

failing to contain Yi agglutinins even in a

dilution of 1 in 5, the minimum Vi diagnostic titre in the serum of a clinically typhoid fever case, was fixed as the 1 in 10 dilution of the serum. A further rise in the Vi titre with the examination of a second sample would establish the diagnosis of the disease.

Fully armed with the proper technique and having secured the necessary information regard- ing minimum diagnostic Yi titre for the local

population, an investigation was carried out on 75 sera of bacteriologically proved typhoid fever cases, as to the presence of Vi agglutinins in

sufficiently diagnostic titre. All the cases were

uninoculated with T.A.B. vaccine. Only one

single sample of blood from each patient was available for testing the serum for the presence of 0, H and Vi agglutinins. The clot culture was done in ox-bile as described previously (Soman, 1932). Thus in every case the clot culture was

positive for B. typhosus. The majority of

samples of blood were received during the second week of fever. The technique of Vi agglutination essentially remained the same as described above except that serum dilutions ranged from 1 in 10 to 1 in 200. In the case of O and H aggluti- nations, the dilution of serum varied from 1 in 50 to 1 in 1,000 and the racks were incubated in the water bath at 56 ?C. for four hours and then were left in the refrigerator overnight. Readings were taken next morning and the results of Vi, O and H agglutinations were com- pared as to their diagnostic significance. The results are summarized in table II.

Table II

O, H and Vi agglutination in the diagnosis of 75 typhoid fever cases

Total number Positive of cases by B. typho- H and O

sits test isolated

70

93%

Positive by

H, O and Vi test

71

95%

Positive by

Vi test onlv

46

61%

Negative by

O, H and Vi test

4

5%

The results clearly indicate the comparative diagnostic value of O, H and Vi agglutination by a single serological examination. Vi agglu- tinins could be demonstrated in sufficiently diagnostic titre in only 61 per cent of the total number of typhoid cases. If reliance was placed on Vi agglutination alone, in 29 out of 75 cases, the diagnosis would have been missed altogether. While by incorporating Vi agglutination test as a routine along with O and H agglutination, only one more case would have been diagnosed. The combined method of O and H agglutination could help the diagnosis in 93 per cent of cases of typhoid fever. In four- cases, no kind of

92 THE INDIAN MEDICAL GAZETTE [Feb., 1945

agglutinins could he demonstrated and the diagnosis was only arrived at by a positive clot culture. Bhatnagar (1938) found the Vi develop- ment to be very regular in 134 cases of typhoid fever, inoculated and uninoculated, and since then many other investigators (Green, 1940; Almon and Stovall, 1940; Eliot, 1940; Coleman, 1942) tried Vi agglutination as a routine test in the

diagnosis of typhoid fever but the results failed to come up to expectations. They could demon- strate the Vi agglutinins in less than 50 per cent of typhoid cases only. Wagle (1941) in

Bombay found the Vi agglutination test to be diagnostic in 67.4 per cent of typhoid cases only, doing a single serological examination. Bensted (1940) found 75 per cent of sera which showed the presence of Vi agglutinins when repeatedly examined at short intervals. Even then in 19 cases he found a complete absence of Vi agglu- tinins in a serum dilution of 1 in 20 or upwards, throughout the disease. Seshadrinathan and Pai (1940) in Madras could diagnose 77.1 per cent of typhoid cases by Vi agglutination test alone. The results of this investigation however showed that Vi agglutination test on a single sample of blood could be of diagnostic value in 61.3 per cent of typhoid cases in Bombay. As the value of any such tests depended on the practical application in the diagnosis of the disease, it was disappointing that the develop- ment of Vi antibody could not be demonstrated in every case of typhoid fever. Under the conditions, the routine combined method of clot culture and O and H agglutination (Soman, 1932, 1934) proved the method of choice for the diagnosis of typhoid fever.

Summary

1. Three hundred samples of sera, sent for Wassermann reaction, were

_

tested for the

presence of typhoid Vi agglutinins. 2. Twenty-five or 8.3 per cent of them only

were shown to contain Vi agglutinins in titres 1 in 5 to 1 in 25. On the basis of these find-

ings, 1 in 10 dilution of a serum was fixed up as the minimum diagnostic Vi titre in a clinical typhoid fever case.

3. Sera from 75 proved typhoid cases were examined for O, H and Vi agglutination, and the results were compared as to their diagnostic value.

4. Vi agglutination test alone could diagnose 61.3 per cent of typhoid cases while 93 per cent of such cases could be diagnosed by the presence of O and H agglutination. In 5 per cent of cases, the diagnosis was solely arrived at by the isolation of B. typhosus from clot

cultures, as none of the three antibodies could be demonstrated.

5. In cases oi typhoid fever, uninoculated with T.A.B. vaccine and with only a single sero- logical examination available, Vi agglutination test, by itself, was found to have hardly any diagnostic value and was actually inferior in

that respect, to the routine combined method of clot culture and 0 and H agglutination.

REFERENCES

Almon, L., and Stovall, J. Lab. and Clin. Med., 25, W. D. (1940). 844.

Bensted, H. J. (1937) .. J. Roy. Army. Med. Corps, 68 1

Idem (1940) .. Ibid., 74, 19.

Bhatnagar, S. S. (1938). Brit. Med. J., ii, 1195.

Bhatnagar, S. S., Free- Indian J. Med. Res., 24, 597. MAN, J. F., and

Dhilon, C. S. (1937). Bhatnagar, S. S., J. Hyg., 38, 663. v

Speechly, C. G. J., and Singh, M. (1938).

Coleman, M. B. (1942). Amer. J. Pub. Health, 32, 843.

Davis, L. J. (1940) .. ./. Hyg., 40, 406. Eliot, C. P. (1940) .. Amer. J. Hyg., Sec. B., 31, 8. Felix, A., and Pitt, Lancet, ii, 186. R. M. (1934).

Green, R. (1940) .. Ann. Rep. hist. Med. Res., Federated Malay States, 1939, Kuala Lumpur, p. 17. Federated Malay States Government Press, Kuala Lumpur.

Horgan, E. S., and Lancet, i, 1084.

Drysdale, A. (1940). Lewin, W. (1938) .. Publications South African

Inst. Med. Res., No. 41, 7, 413.

Pijper, A., and Crocker, ,/. Hyg., 37, 332. C. G. (1937).

Radowsky, H. (1942) .. Trans. Roy. Soc. Trop. Med. and Hyg., 36, 45.

Seshadkinathan, N., and Indian Med. Gaz., 75, 735. Pai, M. N. (1940).

Soman, D. W. (1932) .. Ibid., 67, 15.

Idem (1934) .. Ibid., 69, 572. Wagle, P. M. (1941) .. Ann. Rep. Ilafjkine Inst.,

1939, Bomb ayf p. 45. Government Central Press. Bombaj'.