A ROUTINE COMBINED METHOD OF ' WIDAL REACTION AND CLOT CUL- TURE' FOR THE DIAGNOSIS OF EN- TERIC INFECTIONS

By D. W. SOMAN, m.b., b.s.

Tutor in Bacteriology, Grant Medical College, Bombay

Diagnosis of the enteric group of infections by methods of hsemoculture or by serological tests,

16 THE INDIAN MEDICAL GAZETTE [Jan., 1932

is an everyday event in the routine of a bac-

teriologist's work, but a combined method of serum diagnosis and clot culture does not seem to have been followed as a routine measure. I am indebted to Dr. Dalai, Professor of Bac- teriology, Grant Medical College, for suggesting to me to follow this method which was referred to in the South African Institute of Medical Research, Annual Report 1929. I have tried to follow this method in 70 cases of blood

samples sent for Widal reactions from the different wards of the Sir J. J. and allied hos-

pitals in Bombay. The technique and the

advantages of this method over others, and the results obtained therefrom, form the subject- matter of this article. The routine method of haemoculture followed

here is to inoculate a 100 c.cm. flask of bile broth with 10 c.cm. of whole blood, taken with due aseptic precautions at the bedside of the

patient; the bile broth is incubated for 24 hours at 37?C. and is then tested for the suspected organism. Method of clot culture.?Five cubic centi-

metres of blood is obtained aseptically from the vein of the suspected case of enteric infec- tion and is sent in a sterile test tube for Widal reaction and clot culture. The whole of the serum from the tube is removed with aseptic precautions and part of it is utilized for the

agglutination test for typhoid, and paratyphoid ' A' and ' B' by Dreyer's method. The

remaining serum is stored in a sterile capillary pipette for future use. The clot is then trans- ferred to a sterile tube of ox-bile, which serves as a culture medium. Fresh ox-bile is obtained from a slaughter house, filtered and filled into sterile test tubes, about 10 c.cm. in each. All these tubes are autoclaved for 20 minutes at 120?C. and stored for use. The tube of ox-

bile with a transferred clot, after proper label- ling, is incubated for 24 hours at 37?C.

Usually organisms grow in the medium by that time and are easily made out in a hanging- drop preparation. From this bile tube a sub- culture is made in simple broth, and an agar slope and sugar media are also inoculated. After incubation for a further 24 hours, the broth culture is available for testing the moti- lity of the germ, the ordinary agar growth for staining reactions and for agglutination with

high-titre sera. Sometimes young cultures are

non-agglutinable, and here sugar reactions in such instances will confirm the identity of the organism. The point of interest in doing cultures by

this method is that occasionally after 24 hours in bile medium, no organisms are seen in the hanging-drop preparation nor does any growth occur in simple broth or on the agar slope sub- cultured from the originally-inoculated bile tube. Under these conditions a negative con- clusion should not be drawn but the bile tube should be further incubated for 24 hours and then sub-cultures should be done. I have come

across this finding occasionally during this

investigation and I do not discard the bile tube as sterile or negative, unless it is tested as described above for three successive days. In

spite of strictest attention and regard for

aseptic manipulations and the inhibitory action of bile on the growth of various other organ- isms, occasional contaminations in the bile tube are met with and thus further complicate the work of isolating the organism.

Out of 70 samples of blood tested for Widal reactions and clot cultures 26 turned out to be

positive for enteric infections. Out of 26 positive samples 24 were positive

for typhoid, 2 were positive for paratyphoid ' A and none for paratyphoid

' B '. There were 12 which gave Widal positive and clot

negative. There were 6 which gave Widal

positive and clot positive. There were 5 which

gave Widal negative and clot positive (when no hsemocultures were asked for). There was 1 which gave Widal negative and

clot positive (hsemocultures were done and were positive). There were 2 which gave Widal positive and

clot positive (hsemoculture was asked for and both were positive).

Discussion of the advantages of the combined method over the single method of serum

diagnosis or hcemoculture 1. Serum diagnosis and hsemoculture are

done from the same sample of blood. 2. At times blood sent for the Widal reaction

is negative and hsemoculture of that patient is not asked for; the clot culture turns out to be

positive for one of the enteric group of organ- isms (as shown in the table). This result is

very important for the physician or the medical practitioner who, in the absence of such a

definite and useful finding and with a negative Widal report sent to him, would very likely treat the case as non-enteric in the general ward of the hospital or at home, with the attendant danger of infection to the whole

ward, attendants and staff. From the point of view and interest of the patient, it is equally gratifying to get this finding from the clot

culture as, in its absence, he is denied the most important line of treatment, that is most care- ful and efficient nursing, on which so much

depend his chances of successful recovery. 3. The clot, when cultured, is practically

free from the serum-agglutinins and thus gives a better chance of a successful positive culture; successful cultures from the clot have been

obtained after keeping it in the ice chest for

2 to 3 days. 4. Very often enteric infections run a very

atypical course from the beginning, or sometimes these are followed by complications and re-

lapses; in such cases the patient does not give a proper and correct history of his illness when admitted to the hospital or at home, the attend- ing physician, with all his clinical acumen,

Jan., 1932J WIDAL REACTION & CLOT CULTURE: SOMAN 17

Table

Serial number

1 2 3

9 10 11

Date

28-11-30

4-12-30

16-12-30

11-2-31

18-2-31 1-7-31

3-1-31 28-2-31 21-5-31 4-8-31 10-8-31

Widal test

+ +

H 0 + H + O

+ +

Clot culture

B. typhosus

Sterile

B. typhosus

B. typhosus

B. typhosus

Blood culture

B. typhosus

B. typhosus

Remarks

10th day of fever. 10th ? ? ?

12th ? ? ?

24th Patient died.

Fever 1 month. 18th day of fever. 10th ., ? ?

Sth day of fever. 22nd ? ? ?

24th ? ? ?

9th ? ? ?

15th

Note:?No haemoeulture was asked for by the physician; blood was only sent for Widal reaction

12 13 14

28-11-30 27-2-31 1-7-31

+

+

B. typhosus B. typhosus 10th day of fever. 7th ? ? ?

10th ? ? ?

Note:?Out of 12 hsemocultures done for enteric infections only three were positive

15 16 17 18 19

20 21 22

23 24 25 26

16-12-30 20-1-31 26-1-31 8-2-31 12-2-31 24-2-31

28-2-31

2-4-31

27-6-31 2-7-31

20-7-31 28-7-31

+ + + + Para A + + + +

+ + + + Para A

Sterile 28th day of fever. 15th ? ?

17th ? ? ?

12th ? ? ?

17th ? ? ?

40th ? ? ?

28th ? ? ?

8th ? ? ? Broncho-

pneumonia, patient died. 15th day of fever. 20th ? ? ?

18th ,, ? j,

12th ? ? ?

Footnote:?The Widal reaction is taken to' be positive in a serum dilution of 1 in 50 and higher; and the standardized agglutinin suspensions from the Standards Laboratory, School of Pathology, Oxford, were used.

The day of fever mentioned in the remarks column indicates the day on which the Widal test and clot culture were done.

hesitates between a Widal reaction and blood culture or sometimes asks for both on different

days; and it is at such a juncture that the combined method of clot culture and Widal reaction comes to his rescue. Besides, the

agglutinin titre of the serum in enteric infec- tions is always fluctuating during their course, and as a result of this a single Widal test possesses practically no value. To test the

sample of blood of the same patient twice, thrice or four times and obtain a gradual rise in the agglutinin titre of the serum has more

practical importance. Under the above-men- tioned conditions, even if the Widal test be

negative, clot culture is bound to turn out posi- tive for the causative organism, thus providing an unchallangeable proof as to the nature of the infection.

5. Apart from the difficulties and fallacies in the clinical diagnosis, the serological reaction, even when done under standard conditions, is not free from sources of error, as is made evi- dent from the recent work of Felix and Olitzki

(1928) on two types of antigenic structure, the flagellar or '0 H' and the somatic or

'

0', which react differently with the corresponding agglutinins. These workers have, shown that sometimes H agglutinins of the serum are either not developed or they are absent; while the O agglutinins present in the serum react only with the special

' 0' antigen. This means that

Dreyer's simple- method for the Widal test is further complicated and requires testing the serum with ' 0 ' antigen also. In the words of Gardner (1929)

' when the customary H Widal is negative, as in those cases where no appre- ciable H titre develops or sometimes after

developing, it disappears, an adequately high 0 titre will give the diagnosis of enteric group of infections \ But it is not possible, however, to identify the infecting species by this test, as the ' 0 ' substance is not specific. Here then lies the decided superiority of the combined method over a single hsemoculture or a single Widal test, done in the usual way or modified for testing 0 agglutinins. The non-specificity

18 THE INDIAN MEDICAL GAZETTE [Jan., 1932

of the ' 0 ' substance is the greatest drawback in the diagnosis, except in the inoculated cases, where it is of the greatest importance. The applicability of this combined method

of diagnosis in inoculated cases will then become of very great interest, as Ledingham (1921) has very rightly insisted that no efforts should be spared to establish a diagnosis on this firm foundation and that the results of

agglutination tests, however carefully per-

formed, can never have the same validity as the actual isolation of the causative organism.

6. During this work I have been able to isolate the causal organism from the clot

during all the stages, of the clinical course of enteric infections, and it follows that the general belief that the causative organism can be isolated from the blood only during the first

week, is not borne out by the findings of the clot-culture method. These periods often over- lap and are only classified to get. the maximum chance of obtaining cultures or serum tests

positive. 7. This method is particularly suitable for

private practitioners not attached to the hos- pitals, who cannot afford to keep culture media with them, and who very often have to send blood long distances to a bacteriological labora- tory. They have only to take the blood asep- tically from a vein into a sterile tube (both these conditions must be strictly followed) and despatch it in the most suitable way. The clot can be cultivated even after two to three days in this climate.

8. The last and the most important advan- tage from the bacteriologist's point of view is that he is easily able to collect a large number of different strains of the organism concerned, with different clinical pictures which he may in his spare time study, classify and differen- tiate in their antigenic behaviour. I have been able to collect a dozen strains of B. typhosus by this clot method during the last ten months of my investigation, whereas depending on the hsemocultures asked for by physicians not more than one or two strains were obtained every

year. Besides obtaining various strains, recovery

of the organism from the clot is one of the

indispensable findings to one who is specially concerned with the investigation of determin-

ing the presence or the titre of 0 H and 0

agglutinins of the serum in infected cases of enteric. From the evidence of the few cases

which I have investigated in the light men- tioned above, I suggest that during the course of infection, some correlation exists between the presence of 0 H and O agglutinins in the serum and the presence of the organisms in the blood at the same time, which markedly influences the prognosis of the case.

In conclusion I strongly advocate this com- bined method, considering the simplicity of

technique, the advantages and valuable infor- mation obtained either for the attending

physician or bacteriologist and I hope to find this method given a wider trial in future.

Finally, I am very grateful to Dr. Dalai, Professor of Bacteriology, Grant Medical

College, and the staff of the department of

Bacteriology, without whose co-operation I would nob have been able to do this work.

References

Felix, A., and Olitzki, L. (1928). Journ. Hyg., Vol. XXVIII, p. 55. Gardner, D. M. (1929). Journ. Hyg., Vol. XXVIII,

p. 376.

Lister, Sir Spencer (1929). South African hist. Med. Res., Annual Report.

Topley, W. W. C., and Wilson, G. S. (1929). The Principles of Bacteriology and Immunity. London: Edward Arnold & Co.