ANALYSIS OF THE VIBRIOS

(After Gardner and Venkatraman)

The "Cholera11 group of v iM o s :

Vibrios resembling the true V .cholerae in biochemical reactions and in the "H 1* antigen.

I ----1" 0" antigen,

Subgroup I "0" antigenSubgroup I I , I I I , I V ,V

Non-haemoiytic

True cholera vibrios, at least three sub-types diff erentiated on minor !,0n antigens.

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Haemolytic

EL Qbr vibrios, two or possibly three sub-types diff erentiated on minor "O ’1 antigens.

and VI, and individual races

Mostly haemolytic, Para- cholera, cholera-like and some El Tor vibrios. Sub-types undertermined.

This useful scheme is necessarily tentative.

Cholera carriers.

The chronic carrier state probably does not occur. Of convalescents about 90 per cent, are free of vibrios within 14 days, and about 99 per cent, are free within 28 days.

Transitory carriers (contacts)(Prom various Japanese reports)

Transience of carrier state;Of 112 darriers, 52 were bacteriologically positive once only.Very few were positive after 7 days and only 2 were positive up to the 2 1st day.The agglutination reaction was negative in all cases at first but became fairly strong after 7-8th day.

Latent infection ;Out of 110 carriers (another series), 51 developed cholera.

* InIntermittency of excretion of vibrios by carriers;

Of 37 carriers In another series;

17 were detected at first faeces examination5 f! 11 second ” 11

12 11 11 third " »2 » 1? « fourth " H1 was 11 ” f ifth " " •

Agglutination reaction in carriers;In a series of 84 carriers. 30 per cent, gave agglutination titres above 1/200 (':'s'up to l/pOOO). Highest titres were recorded 4,5, 8 or 9 days after discovery as carrier. The agglutination test was negative in 17 .5 per cent, of the cases soon after cessation of the carrier state. In very transient carriers the agglutination reaction was npgative In 12 days, but it lasted 32 days when the carrier state was more prolonged.

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Enrichment media for isolation of V. cholerae

Dieudonne medium

Sterile, deflbrinated ox blood is collected under aseptic conditions. Equal volumes of the defibrinated blood and normal sodium hydrate solution are mixed and poured into large flasks which should be about half f ille d . The flasks are lightly plugged with cotton wool and the alkaline blood mixture is steamed at 100°C . for thirty minutes. The alkaline blood mixture is then stored at room tempera­ture until it haa matured (this may take from four to eight weeks).

To prepare the solid medium for plates, take three parts of the alkaline blood mixture warmed to 50°G. and add to seven parts of melt>- ed nutrient agar. After thorough mixing, the medium may be poured into sterile Petri dishes and is ready for use when set and dried.

Goldberger8s Alkaline Egg Agar Medium

Contents of one egg (aseptically collected) added to an equal volume of sterile water, and, after thorough mixing, this egg solution added to its own volume of 5 .0 $ solution of sodium carbonate. This mixture is steamed for thirty minutes in a sterilized flask and may then be stored.

To make the solid medium, take five volumes of nutrient agar, containing 1% cane sugar, melted in the steamer and add to it one volume of the alkaline egg solution. The mixture is then tinted with neutral red (one part per hundred of a 0.5% solution). Pour plates.

Prepare as with nutrient broth and set reaction to between pH 8 .0 and pH 9 .0

For v/ater examination this medium may be made up in ten times the above concentration. One volume of the medium is then added to nine volumes of the water under examination.

Goldberger's Alkaline Egg Broth

Alkaline egg solution prepared as for Goldberger’ s alkaline egg agar.

Dunb ar ' s P ep t on o Wat er

PeptoneSodium chloride Sodium nitrate Water

10 gms.5 11

0 .1 i! 1000 ccs.

One volume of this solution is then added to nine volumes of ordinary peptone water. The medium is then tubed and sterilized by steaming. It should be used within a week of making.

Employed in the sub-acute and chronic stages when direct diagnosis by film or culture is of little value.

The antigen

1. A simple suspension of gonococci in saline usually employed, prepared by washing off serum agar slope cultures of several distinct strains and mixing the suspensions, i .e . antigen is polyvalent.Some workers put up the fixation test in duplicate. In one test an antigen prepared from a single predominant type strain is employ­ed while in the other an antigen prepared from one of the inter­mediate type strains is used. In any case the antigen must first ba titrated to determine a suitable dilution for uso.

2. Price 's antigen (Price, I .N .O . L .C .C . monograph No. 2995, 1933) Price has recently described a s p e c i f o r m of antigen which is claimed to be much more sensitive without being less specific.Briefly it consists of a NaOIi extract of the bacterial cells, which is filtered and acidified with HC1. Whito floccules form, which can be centrifuged down, and redissolved In UaOH. This solutionon neutralisation forms a clear solution, which is used as the anti­gen.

The Haemolytic System

The us»A$ anti-sheep cell haemolytic system is employed.

Determination of the strength of the reaction

In this fixation test the strength of the reaction is usually determined by varying the amount of complement present and keeping the other reagents constant. Some workers, however, vary the antigen. The technique varies considerably with different workers, and great care Is nocessary in interpreting the results.

A. The Complement Fixation Test at Different Stages of the Disease when Antigen (1) has been' employed

Acute Gonorrhoea 2-4 weeks 48$+Subacute and Chronic 61%+Epididymitis 82$+Prostatitis 8C$+Visiculitis 88$+Metritis 68/0+Salpingitis 77$>+Vulvo-vaginltis 5 0$+Arthritis 82$+

Points to be noted in interpreting the C .F . test

1 . It is not generally + in the first two weeks, when diagnosis is best done by film and culture.

2. It Is generally negative in simple anterior urethritis and urethritis in the female.

3. It is generally + when complications are present.

4. It remains + for about throe months after clinical cure. A + reaction becoming - at that time is'strong evidence In favour of real cure.

5. A single - reaction is of little importance, "but two or three successive - reactions obtained over a period of 4 to 6 weoks areof considerable importance in excluding the presence of the disease.

6. A + reaction denotes recent activity of the gonococcus in the body.

7. Vaccine therapy invalidates the test for cure, but a rise in titrc is significant in assessing value of vaccine therapy*

8. Reaction practically specific for gonorrhoea (but also + in cerebro-spinal meningitis).

B. The Complement Fixation Test at Different stages of the Disease when Antigen (2) [Price 's] has been employed.

27/iH-in 1st week 46%+ in 2nd week 70fo+ " 3rd "89%+ " 4th "

10Q^+ " 5th "

The Complement Fixation Reaction for the Diagnosis of S7/~philis (Wassermann Reaction)

Employed in all but the earliest stage when direct examination of fluid obtained from primary chancre is more likely to give a posi­tive result.

1. The Antigen. Prepared as follows;A. Heart muscle from ox, calf or man, is extracted with acotone.The acotono-extracted material is dried, powdered and ground up and extracted with absolute alcohol (100 c .c . per 20 gms. of powder). Shaken and allowed to stand for from 2 days to 3 months. Filtered.

B. 1 gm, of pure cholesterol is dissolved in 100 c .c . of absolute alcohol. Before use, 5 parts of solution A are mixed with 3 parts of solution B, and diluted 1/25 in saline. The antigen is then ready for use.

2. The usual anti-3hoop cell haomolytic system is employed.

3. The preliminary complement titration is done in duplicate.

To determine the strength of the Complement Fixation Reaction

(a) A series of dilutions may bo made of the patient’ s serum, the other reagents veing kept constant, e .g . l/2-g, 1 /5 , 1 /10 , 1 /2 0 , 1 /40 , l /8 0 , 1 /160 , or only three dilutions may be put up, e .g . l / 5 , l /l 5 , l /4 5 . This latter method (the three tube method) is usually adopted as a routine. If fixation occurs in the 1/5 dilution the result is reported as doubtful, i f In the l /l5 dilution it is reported as positive, if in the .1/45 dilution, it is reported as strongly positive.

(b) A scries of dilutions may bo mado of the complement, containing e .g . 8 , 6, 4, 2 tho other reagents being kept constant -the patient!s scrurn constant at l /5 . In this method a strongly positive reaction would be indicated by complete fixation of 8 M .H.D. while fixation of 2 M .H .D . only would be reported as doubtful.Whichever method is employed, a patient’ s serum control tube should y/' always be put up, containing the lowest dilution of patient's serum employed in the test, and all tho other reagents except the antigen. This control should show no fixation.

Comparison of Wassermann and Kahn Reactions

1. Wassermann needs more reagents and has more steps than the Kahn,but greater complexity more apparent than real.

2. W.R. less dolicatc than Kahn, especially in treated cases, butoccasionally W.R, + and Kahn - cases do occur.

3. W.R. reverts to negative earlier in treated cases than Kahnand in uncured cases will revert to + less readily than Kahn.

4. W .R. slightly more specific, is more controllable and is lesslikely to give false positives than the Kahn.

It is best then to do both te s ts ,if possible, placing more reliance on Wassermann in primary diagnosis and more on Kahn as test of cure.

Interpretation of rosults

A. In primary'diagnosis(1) Negative. Of little value in excluding in excluding

syphilis by itself sinco at practically no stage is it 1 0 0 . But in conjunction with clinical signs and history may be of value especially if repeated.Percentage of Positive Wassermann Reactions at various stages of disease (Ha r r I son, L ,~W. )

Stage No. of cases i tPrimary 2596 69 .4Secondary untreated 2449 (97 .1

fi all cases 4556 >90.1Latent -untreated 196 72 .9

" all cases 6829 45 .6Tertiary untreated 255 98 .4

” untreated 1829 82. 9Tabes untreated 25 100 .0

" treated 911 66 .0G .P .I 877 97.3

Congenital with manifestsymptoms 394 96 .9

" with latent symptoms 34 82 .3

This table serves to show the comparative value of negative results at various stages, ascertained clinically . N ,B . (i) occasionally-^ patients remain - with extensive lesions. This very liable to happen in (a) myocardial lesions (b) C .N .S . involvement. C .F .S . must be examined in such cases.(ii ) doubtful reactions. Bost settled by giving a small provoca­tive injection of arsenical, and testing 7, 14, and 21 days later.If positive reaction will v e definitely strengthened. Never allude to them as "weakly +'; .( i i i ) positive reactions.TTa^nllvL rh* agnostic of svohilis but., (l) yaws, (2) leprosy (3) trypano­somiasis (4) relapsing fever (5) malaria (6) scarlet fever (7) tropical ulcers may also give undoubted + reactions. In such cases diagnosis must be based mainly on other grounds. Clinically this should not be difficult except in some cases of yaws. Certain

skin diseases probably give + reaction, e .g . psoriasis, erythacma rosa.

B. As an.aid to prognosis in primary syphilis

Three typos of cases.(a)-before treatment starts remains -(b)- " " " becomes +(c)+ " " "

Prognosis for early cure less favourable in that order from (a) to ( c ) .

C. In assessing effect of treatment

Serum reactions of little use at first in primary cases, where treat­ment is commenced with negative serum reaction. ' In primary + reactors clinical signs always disappear before reaction becomes nogative.Negative reactions obtained after course of treatment more significant than when obtained a fortnight or month later, owing to provocative effect of treatment.

D. In determining question of cure

(a) consider doubtful or partial reactions need more treatment.(b) negativo reactors should be tested.

(i) every three months during 1st year(ii ) " six " afterwards, minimum two years,

until satisfied that cure has been effecttial. All tests should be preceded by provocative injection of arsenical to light up any latent infection.

JUCerebro-spinal fluid need only^cxamined(a) later after treatment, a.s assessing cure(b) in latent cases(c) in all cases with C .N .S . symptoms.Amount of fluid in W.R, l /5 to 1 /2 .5 , l / l to 2 /1 . y

Collection Number: AD843

XUMA, A.B., Papers

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