The use of an Onchocerca volvulus microfilarial antigen skin test in an epidemiological survey of onchocerciasis in Guatemala

543 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE

AND HYGEW,

VOL. 73, No. 5, 1979

The use of an Onchocerca vo/vu/us microfilarial epidemiological survey of onchocerciasis YOSHIHISA HASHIGUCHI**, National

antigen skin test in an in Guatemala*

MASATO KAWABATA***, GUILLERMO ZEA F., MANUEL M. RECINOS C. OTTO FLORES C. Department for Malaria Eradication, Ministry of Public Health, Guatemala

Summary Onchocerca volvulus microfilariae obtained from onchocercal nodules were used as antigenic material for a skin test. This antigen is as specific as those previously employed in skin tests, detecting 85 *2”;, of the persons with positive diagnostic signs of onchocerciasis in endemic areas. A low rate of false positives was found in the control group in nonendemic areas. The positive rate of reactions in the persons without positive signs in the endemic areas might be due to the presences of cases not detected as a result of palpation for nodules and two skin biopsies. In the examination of the residents of several populations, a close correlation was found between the positive rates of skin tests and the presence of infection. From these findings, we conclude that the skin test using the microfilarial antigen is a suitable procedure for the diagnosis of onchocerciasis. The skin test could also be useful as an epidemiological tool in assessing the effectiveness of control programmes. Introduction Various antigens have been investigated by many workers for their suitability for the clinical epidemiological diagnosis of onchocerciasis, in the hope of finding a suitable substitute for skin biopsies (KAGAN, 1963). An immunological test could help in the diagnosis of those people recently infected who are difficult to find during epidemiological surveys. It is therefore desirable to standardize a skin test using specific antigens for the diagnosis of onchocerciasis and for evaluating control programmes. Various filarial worms, including Onchocerca vo1vulus, 0. cervicalis, 0. gutturosa, Dirojilaria immitis, Loa loa, Litomosoides carinii, Setaria equina and Contortospiculum rheae have been tested for their immunological specificity against onchocerciasis. Most of these antigens, however, are not specific and cause false negative or false positive reactions. The World Health Organization Expert Committee on onchocerciasis emphasized in their Technical Report Series (1976), the need for the isolation of adequate antigens from adults, microfilariae and infective larvae of 0. VOZVUZUSfor research needs. CIFERRI et al. (1965) worked with 0. volvulus adult antigen and concluded that this antigen produced high positive rates in both healthy and- onchocerciasis-positive persons in endemic areas of the disease. ULRICH et al. (1970) investigated 0. voZvulus microfilarial antigen and obtained-86 :A

AND

positive rates for onchocerciasis patients and 13~4 for control populations with this antigen. In Guatemala, it is possible to obtain many onchocercal nodules as a result of a denodulization campaign which has been carried out in this country since 1935. We have, therefore, used the microfilariae obtained from onchocercal nodules as material for a skin-testing antigen. In the present study, the microfilaria-derived antigen was assessed and the possibility of using larval antigen as an epidemiological tool for the study of Guatemalan onchocerciasis was investigated. Materials and Methods Antigen preparation Onchocercal nodules excised from patients were immediately dissected into several pieces in petri dishes containing saline solution (0.9’;:) and stored at 4°C for 24 to 72 hours. The suspension was then filtered through a sheet of sterilized gauze, for the purpose of removing tissue fragments. The sediment was washed twice with sterilized distilled water and six times with sterilized 0.9 I;{, saline solution, repeating the centrifugation for five minutes at 3.000 r.o.m. The sediment finally obtained was homogenized in a tissue grinder. The mixture was allowed to extract for 72 hours in saline, frozen to -20°C and re-thawed repeatedly, and then centrifuged at 8,000 r.p.m. for 30 min. The amount of protein in the supernatant fluid was determined by the method of LOWRY et al. (1961), and adjusted to 20 pg and/or 10 I-Lg per ml by adding sterile saline. Fracin was added to the antigen as a preservative at the concentration of 0.01 :‘” and the antigen was stored at 4’C until used. Procedure of skin test 0.02 ml of antigen solution was injected intradermally on the forearm using a tuberculin syringe with a 27-gauge needle. Before injection the volar surface of the arm was swabbed with alcohol and allowed to dry. It was confirmed that injection of * This study was supported by the Ministry of Public Health, Republic of Guatemala, and by the Japan International Co-operation Agency (JICA) (GJCRCPO-MENSAP series No. 8) ** Present address: Department of Parasitology, Kochi Medical School, Nangoku City, Kochi, Japan. *** Present address: Department of Parasitology, National Institute of Health, Tokyo, Japan.

544

0.

VOhUh

MICROFILARIAL

saline solution with 0 *01 “/‘o Fracin produced no reaction. The wheal caused by intracutaneous reactions on the forearm were recorded after 15 min in those under 14 years of age and after 20 min in those 15 years old or over, by circling the wheai with a ballpoint pen and transferring the ink marks on to alcohol-moist paper. Later, in the laboratory, the wheal area was measured with a planimeter (Uchida Yoko Co., Cat. No. 906-1500, Tokyo,

Japan). Populations examined Skin tests were carried out on 659 residents in the endemic areas of onchocerciasis and on 106 control persons in Guatemala City who had no history of contact with endemic regions of onchocerciasis. Before examination, people living in the endemic area were questioned about any previous presence of nodules. Two skin biopsies were made by a Holth type sclero-cornea1 punch-one from the left scapula and the other from the iliac crest in males and from both sides of the scapula in females. Subcutaneous nodules were also searched for by palpation. Stools of 222 persons, including the control group, were examined using the method of HUNTER et al. (1948). Preliminary

Results investigation

23 onchocerciasis patients aged 21 to 76 years were tested with the antigen at concentrations of

SKIN

TEST IN GUATEMALA

10 pg and 20 pg per ml. All of these patients had a high density of microfilariae. No significant difference was found in the intracutaneous reactions obtained with the two concentrations (Fig. 1A). The 20 pgjml antigen was used for the epidemiological survey. The relationship between the time elapsing between injection and development of the maximum size of the wheal area was ascertained in the following subjects: 11 males, aged 28 to 62, who had had temporary contact with an endemic area of onchocerciasis; 10 females, aged 19 to 51, with no history of contact with an onchocerciasis region; 24 schoolchildren in the endemic area of Finca Sibaja, Yepocapa; and 29 schoolchildren, aged six to 12 in Guatemala City, a non-endemic area. The maximum size of the wheal area in those 19 years of age or older was recognized 20 min after the antigen injection (Fig. lB), whereas in those less than 12 years old, the wheal reached maximum size 15 min after injection (Fig. 1C). To investigate the influence of age on the reaction, the intracutaneous test was carried out in 68 schoolchildren in endemic and 88 in non-endemic areas of onchocerciasis. The wheal areaincreased gradually with age in the children from endemic areas but no parallel size increase was found in the children from non-endemic areas (Table I). To examine the possible occurrence of cross reactions with intestinal parasites, 75 people from non-endemic areas and 147 from endemic areas were tested (Table II). 39 of the 75 subjects from the former had one or more intestinal helminths (Ascaris-24, Trichuris-26, hookworm--one and tapeworm-one). Three of these 39 individuals had a positive skin test. 55 of the 147 subjects in the endemic areas had demonstrable microfilariae or nodules. 89 y0 of these patients were positive to skin test. The prevalence of intestinal parasites was very high in the endemic areas, 142 of the. 147 subjects investigated having one or more helminths (Ascaris -112, Trichuris-116, hookworm-39 and tapeworm-two). In non-endemic areas, no significant relationship was found between the positivity rates of skin tests and the presence or absenceof intestinal helminths. A comparison of the positivity rates of skin tests in cases with and without mf and/or nodules, showed a significantly higher rate (89 * 1::) in the former than in the latter (50-O 96).

z4:E;!-yI-i$ Application survey

5 Minutes

1o after

15 antigen

20

25

30

injection

Fig. 1. Changes in the mean wheal area with standard error after skin test. A : Reactions against lO&ml (dotted line) and ZO~g/ml (solid line) of protein of the antigen in endemic areas, B : Reactions of adults in cm-&act with the endemic regions (solid line) and those without wntact with endemic regions (dotted line), C : Reactions of schoolchildren in endemic areas (solid line) and those of non-endemic areas (dotted line.)

of the skin test to an epidemiologicaf

On the basis of the results obtained in the preliminary investigation, we applied the skin test to an epidemiological survey of residents in endemic areas of onchocerciasis. For this purpose, 484 subjects in endemic areas and 106 controls in nonendemic areas were tested. In the endemic areas, 136 (28.1 o/o)out of 484 subjects were positive for microfilariae and/or nodules. As shown in Fig. 2, most of the wheal areasof the control group (106 persons) distributed around 20 mm”. Much larger wheal areas were found among the population living in endemic areas, regardless of the presence of mf and/or nodules and we considered reactions positive when the wheal area was 56 mm? or more. No differences in wheal

Table I-Wheal age groups

areas elicited

by 0. volvulus microfilarial

Age in years

antigen

in schoolchildren

Endemic area No. examined

5-6 7-8 9-10 11-12 13-14

545

et a 1.

Y. HASHIGIJCHI

6 22 18 14 8

arranged

Non-endemic

area

Mean & S.D.*

No. examined

Mean :L- S.D.

47.2132.8 50*0+30.4 46.4123.6 68.Ok22.8 67*6+33*6

3 24 28 27 6

25*2_i7 4.8 14.4,k 10.0 21.6i15.6 21.6 j 21.2 28.0-1~ 8.0

by

-

* Standard deviation

)-

Wheal

area

( mm21

Fig. 2. Frequency distribution of wheal areas elicited by skin tests. : Reactions of the control group innon-endemic areas, : Reactions of the persons with mf and’or nodules in an endemic area, - - - - : Reactions of the persons without mf and/or nodules in an endemic area.

areas were recognized among the persons with only mf and those with both mf and nodules. In endemic areas the positivity rate increased gradually with age ranging from 37.5 “5 in those aged nought to four years to 90.9 “b in those 60 years old or over. The frequency distribution of wheal areas in cases with and without mf and/or nodules is shown by sex in Fig. 3. Skin tests with microfilarial antigen

were more frequently positive in male subjects than in females (p < 0.001 in both cases with and without mf and/or nodules). In order to evaluate the antigen as an epidemiological tool, skin tests were performed on the residents of areas with three grades of endemicity, namely, high endemic (Pueblo Palin), medium endemic (Fincas San Rafael Sumatan, Victoria and

546

0.

Table II--Summary among schoolchildren

vohdUS

MICROFILARIAL

SKIN

of stool examinations for helminths in non-endemic and endemic areas

TEST IN GUATEMALA

and intracutaneous tests for of onchocerciasis in Guatemala

onchocerciasis

No. i-ve to skin tests among helminth / ve and mf and/or N -ve

among helminth -ve and mf and/or N -ve

among mf and/or N -I ve

among mf and/or N -ve

(“h)

(%I)

(“;I)

(“:I)

(“
Nofoive

i-ve for mf and/or N* (“/,)

No.

NO.

No. examined 75 (Non-endemic)

i-ve

for helminths

55/147 (37.4)

147 (Endemic)

No. I-ve for helminths helminths No. ~ve among mf among mf and/or to skin and/or tests N -ve N -I ve

(‘:A)

(‘L)

(%)

39/75 (52.0)

5/75 (6.7)

-

39/75 (52.0)

3/39 (7.7)

2136 (5.6)

-

5/75 (6.7)

95/147 (64.6)

51155 (92.7)

91/92 (98.9)

45191 (49.5)

l/l (100.0)

49/55 (89.1)

46/92 (50.0)

142/147 (96.6)

*Nodules

A A

C

20 I 23

24 , 39

LO I 55

56 I 71

Wheat

72 I 87

80 I 103

area

104 120 136 152 168 I I I , I 119 135 151 167 183

( mm2

184 200 I t 199

)

j i.TfiT;;

Fig. 3. Frequency of wheal areas produced by intracutaneous injection of onchocercal antigen in the subjects without mf and/or nodules (A) and with these signs (B). Solid line shows males and dotted line indicates females in an endemic area.

Sibaja) and low endemic areas (Aldea La Cruz and Calderas). Table III shows the reactions found in people with and without signs. The frequency distributions of wheal areas in each population are portrayed in Fig. 4. The results of reactions obtained from the populations in non-endemic areas of onchocerciasis are also shown. It is seen that the frequency of positive tests increased with the intensity of the infection. Discussion

The intracutaneous test with 0. volvulus microfilarial antigen used in this study is as sensitive as

z!E,

,

I 23

{L

4:

5,6

7;

0,0

19'

39

55

71

07

103

119

Wheal

area

l;O 136 152 I , 135 151 167

,

,

168 ML 21% I I I 183 199

( mm21

Fig. 4. Frequency distribution of wheal areas by skin tests arranged by the grade of infection. A : Non-endemic areas, B : Aldea Calderas, C : Aldea La Cruz, D : Fincas San Rafael Sumatan, Victoria and Sibaja, E : Pueblo Palin.

547

Y. HASHIGUCHI et al.

Table III-Infection rates of the residents frequency of positive skin tests

of areas

of varying

Frequency No.

with +ve

+ve

signs

of positive

onchocercal

endemicity

and the

reactions

without

+ve

signs

Statistical significance of the positivity rate in those with and without signs

with mf and/or nodules

(I,,

Aldea Calderas

251175

14.3

18125

(72.0)

301150

(20.0)

x2 29.109 (P
Aldea La Cruz

37,‘239

15.5

30137

(81 .O)

1131202

(55.9)

x2 8.224 (P 10.01)

Fincas San Rafael Sumatan, Victoria and Sibaja

70/105

66.7

59170

(84.3)

25135

(71.4)

2 : 2.411 6. s. f)

Pueblo Palin

26127

96.3

24126

(92.3)

l/l

(100.0)

Locations examined

* Not significant ** By Fisher exact probability

No. +ve/no. exam. (“
No. +ve/no. exam. (“
I’ 0.922** (n. s.)

test

previously reported skin tests, being positive in 85 a2 ‘I;, of the persons with mf and/or nodules in an endemic area of onchocerciasis in Guatemala. In the endemic areas there was a high percentage (56 *4”,) of positivity among the residents who were negative for mf and/or nodules. This finding may represent the presence of subclinical disease and sensitization in the endemic area, rather than a non-specific cross reaction, for positive tests were found in only 7.7 “;) of those with helminths in non-endemic areas. In this study, we employed skin biopsy with two snips and palpation for nodules for diagnosis but these procedures will not detect all cases. Further, the lag of up to six months between the time of infection and the time of appearance of microfilariae in the skin could also contribute a number of undetected infections, as mentioned by MUELLER et al. (1973). The age of the subjects tested influenced the frequency of positive reactions among persons under 14 years of age in endemic areas, as recognized by CIFERRI et al. (1965) who worked with 0. volvulus adult antigen. The sensitivity of those in the lower age group to 0. voZz~uZusmicrofilarial antigen was high in the endemic area of onchocerciasis suggesting that the present antigen is applicable to the evaluation of control programmes, in as much as that it detects recently infected young people. The lower sensitivity of females to the present antigen is of interest. This finding can probably be explained by the differences in the intensity of infections between males and females in the endemic area of Guatemalan onchocerciasis where more

frequent and heavier infections are found in males (82”,,) than in females (20”,,). In Calderas, it was seen that 20.0”,, of the persons without positive findings of onchocerciasis showed a positive reaction, while in La Cruz, 55.9”,, of nononchocerciasis persons reacted positively. The discrepancy could be explained by the differences in the geography and social characteristics of the two populations. In Calderas, no breeding places of blackflies, such as Simulium ochraceum, S. metallicum and S. callidum, were found. Therefore, most of the patients in this village with onchocerciasis probably contracted the disease when they were working in endemic areas as seasonal labourers. On the other hand, there were many breeding places near La Cruz, and the residents were frequently exposed to biting by blackflies. Additionally, in Fincas San Rafael Sumatan, Victoria and Sibaja, those without mf and/or nodules showed a high sensitivity to the antigen, demonstrating a positive rate of 71.4”,. The rate of positive reactions in those without positive signs may, therefore, be a useful indicator of the intensity of infection or sensitization of the population with 0. z~olz’z~lusin the endemic areas. The frequency distribution of wheal areas in the villages mentioned above, was closely correlated with the infection rate of people with 0. vohulus. TADA & OTSUJI (1967) and TADA & FIGUEROA (1969) emphasized the usefulness of skin tests as an epidemiological tool. They recognized the good correlation found between the frequency distribution of wheal size in the reaction to Dirojilaria

548

0.

VOkJU~US MICROFILARIAL

immitis antigen and the infection rate of those with 0. volvulu~ in Guatemala or with Wuchereria bancrofti in Tanan. With our skin test it mav be possible to “as’sess the effectiveness of control programmes for onchocerciasis before and after vector control. ULRICH et al. (1970) reported that the positivity rate as indicated by wheal areas was 7076 in 61 patients with Venezuelan onchocerciasis. Their 0. volvulus microfilarial antigen contained 5 pg of protein, extracted with physiological saline. The rate increased to 86”; when erythema was used to indicate a positive test. In the present study, we used only wheal area as a positive criterion. The microfilarial antigen used in this study, has several advantages over preparations previously used in skin tests: (i) the material, microfilariae, is isolated with ease from onchocercal nodules which are readily obtained in Guatemala; (ii) the antigen can be prepared within a short time as a crude extract using saline solution; and (iii) approximately 20 nodules-supply sufficient antigen-solution for 2.000 tests. More detailed investigations of the present antigen are in progress in our laboratory, with view to its mass-production and to the definition of its antigenic characteristics. Acknowledgements We wish to thank Drs. Isao Tada, Takesumi Yoshimura and Horatio Figueroa M. for their helpful criticism in the preparation of this paper, and we are also grateful to Drs. Tuan Tose Castillo 0. and Hiroshi Tikahashi for their encouragement throughout this study. The present work was carried out with the assistance of our collaborators in Servicio National de Erradicacion de la Malaria, Guatemala, and we should like to thank all those who participated in the field work. We are also indebted to Messrs. 1. Onofre Ochoa and Pedro A. Molina who kindly supervized our field work throughout this study. Mrs. Kazue Hashiguchi helped us to prepare this paper.

SKIN

TEST IN GUATEMALA

References Ciferri, F., Kessel, J. F., Lewis, W. P. & Rieber, S. (1965). Immunologic studies in onchocerciasis and bancroftian filariasis. 1. Intracutaneous test with antigens extracted from Onchocerca and Dirojilaria. American Journal of Tropical Medicine and Hygiene, 14, 263-268. Hunter, G. W. III, Hodges, E. I’., Johnes, W. G., Diamond, L. S. & Ingalls, J. W. Jr. (1948). Studies on schistosomiasis. II. Summary of further studies on methods of recovering eggs of Schistosoma japonicum from stools. United States Army Medical Bulletin, 8, 128-131. Kagan, I. G. (1963). A review of immunologic methods for the diagnosis of filariasis. Journal of Parasitology, 49, 777-798. Lowrv. 0. H.. Rosebroueh. N. T.. Farr. A. L. & Randall, R. k. (1961). &otein measurement with the Folin Phenol Reagent. Journal of Biological Chemistry, 193, 265-275. Mueller, J. C., Mitchell, D. W., Garcia-Manzo, G. A., Aguilar, F. J. & Scholtens, R. G. (1973). Evaluation of a skin test for onchocerciasis in Guatemala. American Journal of Tropical Medicine and Hygiene, 22, 337-342. Tada, I. & Otsuji, Y. (1967). Skin test as a method of epidemiological survey on filarial infection. Japanese Journal of Parasitology, 16, 119-126. Tada, I. & Figueroa, M. H. (1969). Reacciones cutaneas al antigen0 FPT, de Dirofilaria immitis en la onchocercosis humana. Reimpreso de la Revista de1 Colegio Medico de Guatemala, 20, 158-163. Ulrich, M., Pinardi, M. E. & Convit, J. (1970). Immunological reactions in onchocerciasis. Transactions of ;he Royal Society of Tropical Medicine and Hwiene. 64. 111-115. World Hialth’ Organization (1976). Report of a WHO Expert Committee on epidemiology of onchocerciasis. World Health Organization Technical Report Series, No. 597, pp. 53-56. Accepted for publication

5th January,

1979.