Wuchereria bancrofti microfilarial antigen in the diagnosis of human filariasis by skin testM Subrahmanyam1, WK Belokar2
1 Department of Surgery, Medical College, Miraj-416 410, India
2 Department of Surgery, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha-442102, India
Wuchereria bancrofti microfilarial antigen was investigated in skin test on: (1) Microfilaria carriers, (2) Amicrofilaraemic cases from endemic villages with and without intestinal helminths, (3) Cases having apparent symptoms and signs of filariasis. The antigen reacted with specificity in cases having apparent symptoms and signs of filariasis. In microfilaria carriers and amicrofilaraemic individuals from endemic areas no reaction was seen. The diagnostic value o f W. bancrofti microflarial antigen in chronic cases has been discussed.
Except apparent elephantiasis the diagnosis of filariasis in amicrofilaraemic cases remains a clinical assumption. Especially this is true of early hydroceles, epididymo-orchitis, lymphangitis or lymph varix when microfilariae cannot be demonstrated in the peripheral smear at night. Development of a diagnostic test which would be sensitive and easy to perform is of value in these cases. This paper reports the results of a skin test carried out with W. bancrofti microfilarial antigen in patients attending the surgical out patient department of our unit at the Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha which is an endemic zone for filariasis.
The antigen was prepared from W. bancrofti microfilaria collected from microfilaria carriers. These organisms were isolated by Dextraven method by using Dextrose saline and dextraven. The isolated worms were then homogenised in normal saline, The homogenate was sonicated. Proteins were estimated by the method of Lowry et al  and the strength adjusted to 80 µg/ml by the addition of required amount of normal saline. 1:10,000 Merthiolate was added as a preservative and the antigen solution was stored in cold.
The test was performed on the following groups of patients:
Group I:- Microfilaria Carriers.
(Patients in whom microfilaria were demonstrated in the peripheral smear. This also included carriers from villages after mass survey).
Group II:- Amicrofilaraemic Cases. (Those who were not suffering from filariasis but attended our hospital for other diseases.
This group was further subdivided into two:
(a) With helminth positive.
(b) With helminth negative.
Group III:- Chronic cases.
(With apparent signs and symptoms of filariasis).
The Antigenic Test: This was performed by injecting 0.05 ml (4 µg of protein) of the antigen solution intradermally on the volar surface of an arm and an equal amount of merthiolated saline on the opposite forearm. The original wheals (due to antigen and saline) were encircled by a ball pen. The results were read after 15 minutes. The raised indurated areas were also marked with a ball pen. The surrounding erythema was not included in the measurement. The wheal imprints just after injecting the antigen and fifteen minutes later were taken on a butter paper moistened with alcohol. The areas of wheals were determined with graph paper and expressed as mm. 
The results of the skin test with W. bancrofti microfilarial antigen on various groups of persons have been summarised in [Table 1]. For the purpose of classification doubling of the wheal area after 15 minutes was taken as the positive skin test. The ratio of the final wheal area to the initial is referred to as the reaction ratio in the sequel. Merthiolated saline medium did not cause any increase in the wheal area 15 minutes after injection. The reaction ratio was less than 1.2 for all non-reactors.
The antigen reacted specifically in chronic cases whereas in microfilaria carriers and amicrofilaraemic cases the reaction was negative except in two cases in group II (B). The reaction ratio also was 3.52 ± 0.032 in chronic cases (Group III), whereas in all other groups it was less than 2.
Diagnosis of Filariasis due to W. bancrofti and Brugia malayi is dependant on the detection of microfilariae at night.
Hence except in apparent elephantiasis the diagnosis of the disease in amicrofilaraemic cases remains a clinical assumption. This is specially true of hydroceles, epididymo-orchitis, cellulitis of the upper and lower limbs and lymphangitis which are commonly seen in this area.
It is recognised that microfilariae may not be demonstrated in the blood of human cases where any of the following conditions exist:
(1) In cases of single sex infection.
(2) Where living adult males and females are not located in the same lymph channel or gland.
(3) Where there is occlusion of the lymph channels inhabited by fertile female so that microfilariae may not reach the blood.
(4) And when adult filariae are dead already.
In view of these, lymph gland biopsy or the use of antigen by skin test or serological tests to demonstrate the laboratory evidence of infection, becomes imperative as an adjuvant in doubtful clinical entities. The skin test is easy to perform on mass scale, less laborious and time consuming, and the greatest advantage is that the patients are not disturbed during the night and less sophistication is required in performing the test.
Filarial worms like other living helminths are known to stimulate antibody production. Antigens from various filarial worms i.e. Set aria cervi,  W. equinaa Litomosoides carnii were tried to detect human filariasis by skin test but these were found to give equivocal results and the antigens were mostly group specific. The highly purified skin test antigen developed by Sawada et al , from the dog heart worm "Dirofilaria immitis" is claimed to be specific. Chandra et al  tried the antigen from the infective larvae of W. bancrofti in mass survey, and found that the homologus antigen was species specific and was not helpful in amicrofilaraemic doubtful cases. The modification of the skin test by a course of diethyl carbamazine has been discussed by Gonraler  and. Chandra et al  .
In cases of filariasis with apparent signs and symptoms like hydroceles or lymph varix the post operative lymphoedema or lymph scrotum has been significant.  A pre-operative aetiological diagnosis of these lesions may have a significance though it is conjuctural at this stage.
Some false negatives in this group are due to use of diethyl carbamazine in those patients varying from 2 years to 6 months prior to conducting this test. The antigen derived from W. bancrofti larvae on the other hand gave positive test with microfilaria carriers, chronic cases and persons from endemic areas.  The W, bancrofti antigen on the other hand gave positive reaction in chronic cases, where as in the carriers the reaction was significantly negative as was noted in our series. In order to pin point the false positive reactions due to helminths, twelve helminth positive cases were subjected to the test, however none gave the positive reaction. Thus in chronic cases the antigen reacted with specificity. This test is valuable in diagnosing doubtful cases.
However this test has to be performed with purified antigen and with varied protein concentrations.
We are thankful to Dr. M. L. Sharma, Principal and Medical Superintendent and to Dr. Sushila Nayar, Director, Mahatma Gandhi Institute of Medical Sciences for their kind permission to publish this paper.
Our thanks are due to Dr. S. N. Girnikar, of the Filarial Research-cum-Training Centre, Wardha for his constructive criticism.
Thanks are due to Shri Shanmukha Rao for his secretarial assistance.