THE BUFFER PRECIPITATION TEST FOR MALARIA (B. P. T.) ADJUSTED FOR

LARGE-SCALE EXAMINATIONS

By E. K. WOLFF, m.d.

City Microbiologist, Colombo

In a previous paper (Wolff, 1939) I described a new serological test for the diagnosis of chronic malarial infection. The test is based on the precipitation of a protein fraction of the blood serum (euglobulin) by buffer solutions, as it can be shown that normal blood and malarial blood behave differently when mixed with such solutions. If a large set of buffer solutions, covering a range from 5 to 8.4, is used it will be seen that the precipitation in normal sera fades out round about 7.4 whilst the preci- pitation of malarial sera extends much fur- ther towards the alkaline side, and, as a

rule, is of a higher intensity within the

ranges in which normal sera also are precipi- tated. Since, for diagnostic purposes, it has been decided to lay more stress on the exten- sion of the reaction towards the alkaline side than on the differences of intensity, I suggested dispensing with most of the solutions and

simplifying the technique by the selection of two solutions only, namely, pH concentration 7.0 and 7.4. Such procedure seems to be simple

518 THE INDIAN MEDICAL GAZETTE [Sept., 1940

enough, but did not satisfy us fully, as the reading of the 1

abridged '

test is not as easy as the reading of the original test.

An attempt was therefore made to improve on the readability of the abridged test without impairing its reliability and simplicity. The new technique differs from the old one

in so far as a control tube has been introduced, which serves a double purpose. Firstly, it allows us to read by comparison, which is easier than direct reading when we have to deal with slight turbidities; and, secondly, it helps us to detect turbidities of sera which are independent of the puffer effect, and are apt to upset the reading if not detected.

In spite of the addition of a control tube, the test has remained as simple as. before, as one

buffer solution is used instead of the two solu- tions recommended before.

Technique 1. Solutions

(a) Test solution? Stock buffer solution pH 7.7 .. One part Glass-distilled water .. .. Four parts Formalin .. .. .. 0.2 per cent

(b) Control solution? Stock buffer solution pH 7.0 .. One part Glass-distilled water .. .. Four parts Normal saline .. .. Five parts Formalin .. .. .. 0.2 per cent

The stock buffer solution pH 7.0 and 7.7 may be prepared either from Baird and Tatlock's Universal buffer mixture or by mixing acid potassium phosphate with sodium hydroxide according to Clark and Lub's formula for the preparation of phosphate buffers. These stock solutions have to be tested before use. They keep even in a hot climate for a considerable time if 0.2 per cent of formalin is added.

2. Blood

The blood should be taken by venipuncture, and, if possible, in the morning before break- fast. About 2 c.cm. of blood is sufficient. It has to be placed in a dry and clean test tube or centrifuge tube. After clotting, the clot is loosened carefully with a glass rod from the wall of the tube, and the sample put in the refrigerator (or a cool place) overnight. The next morning the serum is ready for the test if well separated and clear; if badly separated or turbid gentle centrifuging may be done. The serum is not removed from the clot till it is added to the solutions during the actual test.

3. The actual test

Test tubes 4 inches^ by half an inch are placed in two rows in a rack, the front row for the test solution and the back row for the control solu- tion. For each serum one tube of each solu- tion is required so that each row contains as

many tubes as the number of sera to be examined. The solutions are distributed over

these tubes in quantities of 1 c.cm. for each tube. One graduated 5 c.cm. pipette may be

used without harm for the distribution of both

the solutions. About 4 drops of serum are removed from the

blood sample and two drops placed in each of the two test tubes. The most practical are

Dreyer's drop pipettes with rubber teats. The

same pipette can be used for all the samples if

carefully rinsed with distilled water after taking each sample.

After the addition of the serum the tubes are shaken and left standing on the table at room temperature.

4. The. reading of the test

The reading may be done from half an hour to two hours after the completion of the test. The average positive case can be read almost

immediately after the addition of the serum; but weakly positive cases develop slower, and for this reason it is advisable to wait 30 minutes before attempting the reading. Usually very small changes take place between 30 and 120

minutes, and there is no need to be specially careful as regards the exact time of reading. The reading is best done by daylight. The

two tubes belonging to the same sample are

held side by side against the window, part of the window-frame providing a background. The light should not be too bright. Artificial light can be used instead, but care has to be taken that the light falls from a higher level from behind on to the tubes. The reading is based on the comparison of

the test tube and the control tube, and, in an average case, should present no difficulties. In most cases the control tube is quite clear and therefore even a delicate turbidity in the other will show up very well. Originally turbid sera show some turbidity in the control tubes, and the difference of the intensity of the turbidities has to be taken into account. Occasionally very strongly positive sera (hyperflocculating sera) tend to precipitate in the control tube to some extent, but always considerably less than in the actual test tube.

^

It is difficult to give hard and fast rules for the recording of the results. I suggested for the original test the following classification : '

opalescent', '

faintly cloudy ', '

cloudy ' and

'

markedly cloudy ', or '

plus-minus ' one plus ',

' two plus ' and '

three plus For the modified test it may be found more convenient to call the

very faint reaction (i.e., a turbidity just notice- able when compared with a clear control) ' doubtful' and the following grades of turbidity '

weakly positive ', '

positive ', '

strongly posi- tive ' and '

hyperflocculating \

Discussion

1. Limitations

Generally speaking, the simplified B. P. T. has the same limitations as the other serological tests for malaria described so far (Tyagaraja, 1938). This is bound to be the case as the

Sept., 1940] DIFFERENTIATION OF BACTERIA FOUND IN WATER : NEOGI 519

leaction cannot claim to be 1specificInci- entally, no such claim can be made for any of lie serological tests for syphilis, which never-

T ess are undoubtedly of the greatest value,

n addition to the lack of '

specificity which may occasionally interfere with the correct variation of the positive readings, there are some difficulties in the reading of the test to be expected when we are dealing with

'

boundary cases . The reaction may become so weak that doubts must arise whether we should call it 1 r ?Tei , ?r s^e^er under the term doubtful . This again is a well-known experi-

ence with all kinds of serological tests. On the other hand, difficulties arising out of such doubts may often be overcome by repeating the test at a later date.

2. Usefulness In spite of these limitations, the usefulness of

these tests seems to me well established. There is only one other disease that gives us positive results so regularly as malaria, namely, kala- azar, but, as a rule, the reaction in kala-azar is very much stronger, and a 'typical' malarial and a typical' kala-azar reaction differ con-

siderably. The '

typical ' kala-azar reaction is the hyperflocculation, i.e., the type of reaction in which a heavy precipitation forms almost instantaneously and settles quickly at the bottom of the tube; whilst the '

typical' malarial reaction does not show this very striking pheno- menon. Still, we may come across such a re- action occasionally in malaria also (and pos- sibly 111 some advanced condition of disturbed liver function); and in such cases an attempt should be made to eliminate or verify the diag- nosis of kala-azar by the performance of the lormol-gel test. In Ceylon, where kala-azar is unknown, this problem does not arise, and we nave to pay attention only to the possible inter- ference by liver disturbances when we come

across '

hyperflocculation \ The value of negative tests seems to be

extremely high, provided, firstly, that the result 1S e?n^rined by a second test at a later date, and, secondly, that the blood is taken at the right time. The acute feverish period of a

malarial attack has to be avoided, as we know that such an attack considerably diminishes the strength of the reaction, and reduces it even to nil if it has not reached a certain height before the test.

Summary The simplified B. P. T. for malaria is per-

formed with one test solution and a control solution. The test solution is a diluted stock buffer

solution pH 7.7. The control solution is a diluted buffer stock

solution pH 7 with the addition of normal saline.

(Continued at foot of next column)

(Continued from 'previous column)

The test takes very little time, and no special outfit is required. It is kept at room tempera- ture and may be read within half an hour. The usefulness of the test is almost the same

as of the original B. P. T. Its simplicity makes it more suitable for routine work on a large scale.

References

Tyagaraja, S. (1938). Journ. Ceylon Branch Brit. Med. Assoc., Vol. XXXV, p. 342.

Wolff, E. K. (1939). Trans. Roy. Soc. Trop. Med. and Hyg., Vol. XXXII, p. 707.