SEROLOGICAL STUDY OF MENINGOCOCCUS

by M. P. Vora, M.B.B.S.

The Medical Bulletin

Number. 9 of Volume No – IX., 1941, Number. 213 of May 3 rd, 1941.

Page No. 288 to 292.

The main object in undertaking this investigation was to ascertain the type of the meningococcus prevalent in the epidemic of meningococcal meningitis, which was raging in the city of Bombay in 1936-37. The problem of different types of meningococci in different epidemics and at different places has been studied to some extent. But the work of this nature and its references are scanty in India. It would appear from those references that the strains of meningococci responsible for the outbreak of Cerebrospinal Fever, irrespective of time and place, are usually the same, i.e. Types III. & I or Group I. This is true not only in India but also in other countries, F. P. MacKie (1923) investigated some cultures here and sent some emulsions of cultures to the Lister Institute, London for further study. The result of his investigation showed that the most prevalent type of the meningococcus then was the type II.

M. L. Ahuja and N. Sing (1935) typed thirteen strains of menigococci, isolated from Delhi and Kasauli, and found that the eleven strains belonged to the type III and the rest to the type I. They confirmed their results by sending the cultures of the same strains to the Pathological Laboratory of the Ministry of Health, London.

They also typed nineteen strains of meningococci isolated from Bombay Presidency; of these, fourteen belonged to the type III and five belonged to type I.

During my stay as a Resident Medical Officer at the City Fever Hospitals, Arthur Road, Bombay, the study of the cerebrospinal fluids of the indoor patients were undertaken and an attempt was made for serological differentiation by agglutination tests of the various strains of meningococci grown from those fluids. The result of the work done at the above Hospital as well as in the Haffkine Institute, Bombay, showed that the types I and III or Group I of the meningococcus was the most prevalent then. Out of 96 strains of meningococci which were typed 84 belonged to Group I.

The precise procedure followed in typing is described briefly in the following few lines:-

Culture of meningococci - A few c.c. of cerebrospinal fluid from the cases of meningococcal meningitis were collected aseptically in sterile test tubes. Fluid was then incubated at 37°C for half an hour to one hour. Primary cultures were done on “Influenza medium” (heated blood- agar – Haffkin Institute). The amount of fluid necessary for culture depended on the nature of the fluid; thicker the fluid, lesser the quantity that was required for culture. Primary cultures grew after 36 to 48 hours incubation at 37°C. Subsequent cultures were done on blood-agar. It was noticed that the primary growth of meningococci from cerebrospinal fluid was easier and more prolific on Influenza medium than on ordinary blood agar and that the former medium was not suitable for stocking the strains and that rabbit’s blood-agar was superior in this respect. Because of the delicacy and fear of dying of the organism, sub-cultures had to be done every third or fourth day.

Identification of organism :-

• Smear of the fluid – Gram-negative intra- and extra-cellular diplococci.
• Culture, morphology, staining reactions, chemical reactions and agglutination.

Since the culture were done from the cerebrospinal fluids, the identification of the organism usually presented little difficulty. Finding of a Gram-negative, intra-cellular diplococcus which fermented glucose and maltose with the formation of acid but no gas, and which did not ferment sacchrose, was practically conclusive.

Method- Gordon and Murray’s four types of menigococci are very definite in their mode of agglutination with the corresponding monovalent sera, and it is also proved and recognised by all workers that a definite relationship exists between type I and type III, and between type II and type IV, and cross-agglutination between them is possible. Moreover, the difficulty of getting the typed organism to prepare the type-sera or the difficulty of obtaining the four-types of high-titre sera to type our organisms, could not be avoided and it proved a hinderance. In view of these considerations a modified method of typing of organisms, which has come into vogue, was adopted. According to this method, we believe in typing or to say it more scientifically, grouping them into two; group I in this case includes Gordon and Murray’s types I and III, and group II in this case includes Gordon and Murray’s types II and IV.

Agglutination technique and necessities:- Macroscopic technique was adopted for the purpose and measurements were made volumetrically.

• Agglutinable suspension of the cocci- the suspension of young meningococcal cultures. 24 to 48 hours old, in normal saline was prepared to the standard opacity tube No. IV. Organisms were then killed by heating the suspension in a water-bath at 55°C for half an hour. It was essential to exclude haemoglobin while preparing an emulsion; for it often interferes with the result of the test.
• Antisera- High Titre Sera Groups I and II, prepared by Standard Laboratories, Oxford; Titre 1 in 250. They were diluted with normal saline to 1 in 50, the final dilution becoming 1 in 100. For economy, ten suspensions were typed at a time.
• Controls- (i) Normal horse serum diluted with physiological saline to 1 in 50, and (ii) Normal saline.
• Rack holding 40, 1 c.c. agglutination tubes, flour rows, each row containing ten tubes, ppts, etc.

Agglutination Test:- To the first tube in every row was added 0.4 c.c. of dead emulsion of the first strain of meningococcus. 0.4 c.c. of diluted High Titre serum group I was added to the first tube in the first row. To the 1 st tube in the second row was added 0.4 c.c. of diluted High Titre serum group II. 0.4 c.c. of diluted Horse serum was added to the first tube in the third row, and 0.44 c.c. of normal saline was added to the first tube in the 4 th row. The last two tubes held as controls. In this way all the remaining strains of meningococci were dealt with successively. This being done the rack was kept in the water-bath at 55°C for four hours, and the results were then read and confirmed by reading them once more after 24 hours. The test was considered positive where there was precipitate at the bottom of the tube and clearing of the upper fluid; control must not show any change. The results were discarded where controls showed any changes.

Results- (The results were submitted to the Indian Science Congress at Karachi Session, 1937).

In all 114 cases were studied –

Successful culture – 96

Unsuccessful cultures – 8

Severe contamination – 6

Cultures died - 4

Out of these 96 successful cultures –

Strongly positive to group I or

types I & III 72

Weakly positive to group I or 84

types I & III 12

Weakly positive to group II or

types II & IV 2

Weakly positive to both groups 2

Not positive to any group 7

Self-aggultinating 1

It is evident from the foregoing results that the Group I (i.e. Types III & I) of the meningococcus was predominant to the extent of 87½ %, in the last out-break of meningococcal meningitis in Bombay.

Acknowledgements:- My thanks are due to Lt. Col. R. C. Wats, M.D., D.P.H., D.T.M., the Officiating Director, Haffkin Institute, Bombay, for kindly allowing me to work in the Institute and assisting me generously in carrying out this work, and to Mr. C. S. Patel, F.R.C.S. (Eng.) the Officiating Superintendent, the City Fever Hospitals and Maratha Tuberculosis Hospital, Bombay, for permitting me to go through the hospital records and to publish the results.

References:

FLEXNER, Journ. Exp. Medicine. XCII, 553.

M. L. AHUJA & N. SINGH, Indian Journ. Med. Resch., 4-4-1935.

RUSSEL, Office International d’Hygiene Public, 1933.

P. T. PATEL, Lancet, 11,539, 1926.

MACKIE F. P., Hospital Records & Reports.

System of Bacteriology, Vol. II.

Medical Research Council Pamphlets on Meningococcal Meningitis, 97-98.

The City Fever Hospitals Annual Reports, 1921-26 and 1936.