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Distribution of microfilariae in Guatemalans with onchocerciasis

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Page 1: Distribution of microfilariae in Guatemalans with onchocerciasis

254

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 77, No. 2, 254-258 (1983)

Distribution of microfilariae in Guatemalans with onchocerciasis

A. DAVID BRANDLING-BENNE?T* AND RICHARD F. DARSIE** Central America Research Station, Center for Infectious Diseases, Centers for Disease Control, Public Health

Service, U.S. Department of Health and Human Services, San Salvador, El Salvador

Summary Ten Simulium ochraceum were allowed to feed at 10 different sites on 12 Guatemalans with

onchocerciasis, and skin snips were taken from six of these sites. Numbers of microfilariae (mff) ingested by the flies and mff emerging from skin snips were highly correlated and showed that concentrations were greatest on the torso and decreased Derioherallv. S. ochruceum ingested the number of mff present in 1.0 mg or under 15 mm2 of skin. Numbers of mff in skin snips from the head, shoulder and upper arm correlated with over-all levels of infection but were frequently negative in subjects with light infections. Two or more skin snips were best able to detect and quantify infections.

Introduction Knowledge of the patterns of distribution of

microfilariae in the skin of humans infected with Onchocerca volvulus is important in diagnosing onchocerciasis and in understanding its transmission. The skin snip provides a sensitive, quantitative and practical means of diagnosing infection with Onchocerca vo1vulu.s. In recent years, methods for obtaining and processing skin snips have improved (COLLINS et al., 1980). However, the sites for taking snips most accurately to detect and measure infections in Guatemalan subjects are not known. Few studies of the distribution of microfilariae (mff) in the skin of Guatemalans have been done (DE LEON & DUKE, 1966; WOODRUFF et al., 1966). The relationship between the mf density in skin snips taken at one or more sites and the over-all level of onchocercal infection must be known if the skin snip is to be used to detect light infections and to measure infections quantitatively.

Since mff are not distributed randomlv in the skin, a larger sample is needed than can be obiained with a limited number of skin snips, if the distribution of mff is to be described in detail. GOLDMAN (1944) suggested the use of black flies for the xenodiagnosis of onchocerciases. DE LEON & DUKE (1966) permit- ted Simulium ochraceum to feed on four Guatemalans and used the uptake of mff by the flies to assess the distribution of mff in the skin. Their methods provided a means of sampling a large number of sites on the human body, while subjecting the participants to minimum risk and discomfort.

In the present study, we first determined the relationshiD between the number of mff found in a skin snip *and the number of mff ingested bv S. ochruceuin at the same site. An earlier study done in Guatemala had found this relationshiD to be nearlv linear (CAMPBELL et al., 1980). We then used thk ingestion of mff by S. ochraceum to determine the distribution of mff in the skin, to assess the total mff load, and to identify those sites which would best indicate the level of infection in Guatemalan subjects.

Materials and Methods The study was carried out on a plantation in the

Department of Suchitepequez, Guatemala, on the Pacific slope of the volcanic mountain range south of Lake Atitlan.

i2 infected men were selected for the study from all plantation residents who had previously participated in a skin snip survey (COLLINS et al., 1980). The subjects were chosen io repreHe& levels of infiction &ging fro& light to heavy. The body of each subject was systematically divided into 10 sites: head (above the posterior hair line); neck and shoulder (above the scapula and clavicle); chest and abdo- men (divided at the lower margin of the sternum); upper back and lower back (in line with the division of chest and abdomen); upper arm and lower arm (divided at the elbow); and upper leg and lower leg (divided at the knee). Only the right or left half of the body was studied, with the choice of side alternating between subjects.

Ten S. ochracacm were allowed to feed on each site of the body. The sites were exposed in sequence, starting with the head and arms, leaving the other parts clothed or draped. Wild flies were allowed to land and, when nearly engorged, a plastic tube, coded for subject and site, was placed over each fly, attempting not to disturb the f ly during feeding. Flies were permitted to release freely once fully engorged. Partially fed S. ochraceum and other species of black-flies were not allowed to feed on the subjects. All collections ofS. ochraceum were made between 7 and 11 a.m., when the parity rate and the risk of transmitting third-stage larvae is the lowest (COLLINS et al., 1981).

Within 30 min after feeding, the abdomen of the f ly was dissected and the gut was washed into the well of a microtitre plate containing NCTC 135 in Hank’s basic salt solution including antimicrobials (COLLINS et al:, 1980). Plates were sealed and, later in the same day the fled was removed from each well, placed on a slide and the mff were counted. The wells were checked under a dissecting microscope for remaining mff.

Immediately after the f ly collection was completed, six skin snips were taken with a Holth-type corneoscleral punch from the following locations on each subject: one cm behind the outer canthus of the left eye, the mid-shoulder, the mid-dorsal aspect of the upper arm, the mid-lower back, the lateral aspect of the mid-thigh and the lateral aspect of the mid-calf. These locations were at the centre of six of the 10 sites on which S. ochraceum were previously allowed to feed. Skin snips were placed in microtitre plates containing NCTC 135 in Hank’s base with antimicrobials and were incubated at room temperature for 24 hours. The mff that had emerged from each snip were then counted. Skin snips were removed and preserved in 10% formalin.

*Present address: Epidemiology Programme Office, Centers for Disease Control. Atlanta. Ga30333. USA. **Present address: Vectorborne Viral Diseases Division, Centers for Disease Control, Atlanta, GA 30333, USA.

Page 2: Distribution of microfilariae in Guatemalans with onchocerciasis

A. D. BRANDLING-BENNETT AND R. F. DARSIE 255

Preserved skin snips were blotted dry and weighed to an accuracy of 0.1 mg. The outline of the superior surface of each snip was then traced with a camera lucida, and the area of the outline was measured with a planimeter.

The logarithmic mean number of mff ingested by S. ochruceum was calculated for each site and each subject using formula

I: In. (x; + n n >

where xi was the number of mff ingested and n was the number of engorged Bies examined. The logarithmic trans- formation of the number of mff per mg of skin snip was obtained with the formula

where xi was the number of mf emerging and wt, was the weight of the snip. A similar formula was used to obtain the mff per mm2 and the mff counts not adjusted for weight or area. All calculations, including means, standard deviations

and linear regression were performed on the logarithmic values. Student’s t test was used to obtain the P values of correlation coefficients (ARMITAGE, 1971).

Results Table I lists for each subject the geometric mean

number of mff ingested by S. ochraceum feeding at 10 different body sites and the geometric mean ingested from all sites. The distribution of mff was centripetal, with the highest numbers ingested from the torso and the lowest from the legs. With increasing levels of infection, the number ingested from the head, arms and torso rose, but the number from the legs remained low. No one site on the torso consistently yielded the most mff.

Table II lists for each subject the number of mff per mg emerging from skin snips taken from six different sites. Similar distributions were seen when skin snips were not adjusted for size, i.e., mff count alone was used, or when skin snips were adjusted for area, i.e., mff per mm2 was used. Skin snips from the lower

Table I-Geometric mean number of microlilariae ingested by Simulium ochraceum feeding at 10 diierent body sites on 12 Guatemahn subjects

Site ABCDEFGHIJKL Overall meant

Head 0.3 0.0 0.3 3.4 0.8 3.7 1.8 13.9 21.3 16.6 9.9 22.1 4.0 Shoulder 0.3 0.0 0.8 6.1 5.9 17.5 3.2 30.6 20.5 39.6 110.1 53.5 9.4 Upper - 0.1 0.0 1.4 2.0 0.0 10.9 32.0 8.9 60.7 21.3 356.8 1515 9.8 Lower arm 0.1 0.0 I.6 1-o 0.1 0.0 18.2 1.8 10.5 O-8 748 125.8 3.5 Upper back 0.7 0.8 6.3 1.6 44.3 53.3 15.6 28.0 26.1 251.7 110.6 136.9 20.0 Chest 0.2 0.5 0.8 7.1 1.0 28.8 49.7 26.3 57.9 39.0 72.2 132.8 12.9 Lower back 5.3 44.6 7-l 14.9 9.7 67 12.3 58.9 12.3 435.0 91.7 69.8 24.9 Abdomen 3.8 28.1 3.8 0.8 6.3 38.0 82.2 42.5 66.0 101.8 148.9 140.9 26.5 Upper leg 0.3 2.3 5.5 0.1 48 4.4 0.4 7.3 1.2 1.6 2.2 8.1 2.3 Lower leg 0.2 0.5 1.9 0.7 9.8 2.1 0.3 1.8 0.2 0.9 3.3 4.9 1.5

Geometric mean number of microfilariae ingested by S. ochraceum from each subject

mean 0.7 l-7 2.3 2.4 3.8 8.8 9.3 14.1 15.3 23.1 45.9 53.7 8.2 All sites

cv* (%) 147.7 153.9 79.8 97.8 88.7 65.3 70.5 53.7 56.5 65.3 47.6 37.2 82.5

*cv = coefficient of variation +calculated as the geometric mean of all microfilariae ingested per f ly at all sites

Table II-Number of microlihriae emerging per mg of skin snip taken from 6 diierent body sites on 12 Guatemalan subjects

Site

Number of micro6lariae emerging per mg of skin from each subject Overall

ABCDEFGHIJKL mean

Head 0.0 0.5 0.0 0.0 0.0 5-O 6.9 7.9 28.8 14.0 3.3 28.2 3.3 Shoulder 0.0 0.6 0.0 2.3 2.3 97.3 5.7 79.2 37.5 31.3 96.7 172.3 11.8 Upper - 0.0 0.0 0.7 o-0 2.0 20.0 102.8 49.3 149.2 10.0 197.9 222.0 12.6 Lower back 5.3 56.4 20.0 16.4 130.6 30.0 142.5 124.1 54.3 355.7 45.6 86.3 54.1 Upper le.2 0.0 2.9 23.8 1.1 8.7 9.3 1.4 3.5 2.9 5.7 20.7 21.7 5.1 Lower leg 1.7 0.0 12.0 1.0 17.1 6.9 6.3 2.9 2.5 0.0 0.7 17.2 3.1

mean O-5 l-9 3.7 1.5 3.8 16.8 14.5 19.7 21.5 14.3 21.8 57.7 9.0 All sites

cv* (%) 177.2 147.4 99.4 116.3 83.1 36.1 60.4 50.5 48.4 70.9 58.8 26.6 74.7

*CV = coefficient of variation

Page 3: Distribution of microfilariae in Guatemalans with onchocerciasis

256 DISTRIBUTION OF MICROFILARIAE IN GUATEMALANS

.

.

Table IlLCorrelation in In, microfilarial count per mg of skin snip taken from different body sites witb In, mean microfilariae (mfQ ingested by S. ochraceum at all sites on 12 Guatemalan subjects.

Correlation coefficient

Skin Snip site r

Head 0.83

Shoulder o-91 Upper arm 0.90

Lower back 0.58 Upper leg 0.54

Lower leg 0.10 Shoulder and lower back (mean) 0.92

All sites 0.93

Note: r3058, ~~0.05 rs0.71, p<O*Ol r30.82, pSO.001

I ‘-;‘.?9,-i-. , ,I, , II1 1 23 5 10 20 30 50 100 200300 500

Mean number (x + 1) of mf ingested

Note: Numerals adjacent to poy7ts represent the number of observations with corresponding values.

Fig. 1. Correlation of the number of microfilariae (mff) per mg emerging from 72 skin snips (x + 1) with the geometric mean number of mff ingested by 10 S. ochraceum (x + 1) at the same site. Line of regression is y = 1.42 + 1.01x, r = 0.87.

back contained the most mff in nine subjects; snips from the upper arm contained the most in three, two of whom had the heaviest infections.

The correlation between the number of mff in- gested by S. ochraceum and the number of mff emerging per mg of skin for the 72 sites from which snips were taken (Fig. 1) was highly significant (r = O-87, p<10m9), and the regression line was parallel to the line of identity. Therefore, for each site, the number of mff ingested by S. ochraceum and the number of mff emerging from the skin snip were comparable. S. ochraceum ingested approximately the number of mff present in 1.0 mg of skin.

The correlation between the number of mff emerg- ing per mm2 of skin and mff ingested was also significant (r = 0.88) and indicated that S. ochraceum ingested the number of mff covered by approximately 1.5 mm2 of skin.

To determine which skin-snip site or sites provided the best measure of infection in each subject, we used mff ingested by S. ochraceum. We accepted this as the over-all measure of ingestion. Correlations were obtained between the geometric mean number of mff ingested and the mff emerging at individual skin snip sites and combinations of sites (Table III). The same correlations were made with no adjustment for skin snip size, i.e., mff count alone, and with adjustment for skin snip area. Correlations were similar, e.g., at the shoulder, r = 0.90 for the unadjusted mff count and r = 0.89 for mff per mm2. Correlations were greatest for combinations of the head, arm or shoulder with one site on the torso. No particular combination gave a significantly better correlation than the others. The relationship of the mean number of mff emerging from skin snips at the shoulder and lower back with over-all level of infection is shown in Fig. 2.

Correlation with In, mean mf ingested

Regression coefficients

a b

-7 -0.71 -0.92 156 -1.27 1.75

2.76 0.56 0.72 0.49

1.20 0.09

0.92 1.06 0.16 0.93

200 1 3, . loo- . l

Eli: c=

/ .

.’

5 b 50 -

.

.; 4 30- .

-= ES 20- .

'i;, $3 'O-

.

3

2%

cE

8' gz

3

/ .

5 .

l 2-

Mean number of mf ingested at all sites

Fig. 2. Correlation of the geometric mean number of microfdariae (mff) per mg emerging from skin snips at the shoulder and lower back with the geometric mean number of mff ingested by S. ochraceum at alJ sites. Line of regression is y = 2.51 + 1.06x, r = 0.92.

We also looked at the correlation between the mean number of mff ingested at a site and the over-all level of infection, i.e., the mean number of mff ingested at all sites (Table IV). The correlations were similar to those obtained by skin snips at six of the sites. The mean numbers of rnff ingested at the remaining four sites (lower arm, upper back, chest and abdomen) also correlated significantly with the over-all level of infection.

Discussion Since S. ochraceum has not been colonized and will

rarely take a blood meal after capture, only wild flies

Page 4: Distribution of microfilariae in Guatemalans with onchocerciasis

A. D. BRANDLING-BENNETT AND R. F. DARSIE 257

Table IV-Correlation of In, mean microfilariae (n&f) ingested at different body sites with In. mean mf ingested by S. ochruceum at all sites of 12 Guatemalan subjects

by S. ochraceum correlated closely with the number of mff emerging from skin snips taken at the same sites, and the variances in these numbers were similar. Mff ingestion by S. ochraceum, therefore, provided us with a better means of quantifying onchocercal infections, since we could examine more sites by xenodiagnosis.

Correlation with In, mean mf ingested

Correlation Regression

Site of coefficient coefficients

ingestion r a b

Head 0.87 -0.42 0.91 Shoulder 0.92 -0.45 1.25 Upper arm 0.93 -1.27 1.64 Lower arm 0.77 -1.09 1.16 Upper back 0.88 O*ll 1.32 Chest 0.90 -0.34 1.30 Lower back 0.65 1.62 0.73 Abdomen 0.86 0.80 1.13 Upper leg 0.36 0.69 0.23 Lower leg 0.33 0.50 0.20 Shoulder and lower back (mean) 0.89 0.59 0.99

Note: rs0.58, ~~0.05 rSO.71, psO.01 ra0.82, p<O.OOl

seeking a blood meal can be used for the xenodiagno- sis of onchocerciases. This procedure poses several potential problems: variation in the size of the blood meal taken, the existence of mff in the gut of the fly before feeding, and migration of mff out of the gut before dissection.

S. ochruceum is not easily disturbed once feeding has begun and will usually engorge fully. After completing the present study, we weighed 100 flies captured before feeding and another 100 cantured afier engorgement. The average weight increased from 0.6 -t 0.2 mg to 1.9 * 0.4 mg, suggesting that flies increst about 1.3 me of blood. Variations in the amount of blood taken-did not appear to interfere with the design of the study. Feeding was done during the same four-hour period each day in order to reduce the influence of the feeding period on the number of mff ingested (TADA & FIGUEROA MARROQUIN, 19741. -- It.is unlikely that mff were present in the wild flies before feeding. Microfilariae from ureviouslv com- pleted feedings would have passed out of thhgut or been digested. Partially fed flies, which might have had mff in the gut, were not permitted to bite subjects during the collections.

In the 30 min or less between engorgement and dissection of the flies, fewer than 1% of mff would have migrated out of the gut (OMAR & GARMS, 1975); actually, less than 2% of ingested mff leave the gut at any time (COLLINS et aE., 1977). Therefore, we felt justified in limiting dissections to the gut. which simplified examination of the 1200 flies.

The skin snip provides a reliable means of measur- ing onchocercal infection at the site where the snip is taken. About 75% of mff will emerge from a skin snip incubated in isotonic saline or NCTC 135 (Hank’s base) for 24 hours (COLLINS et al., 1989; SCHULTZ- KEY, 1978). In our studv, the number of mff ingested

Our results do not support the contention made by others that Simulium species concentrate or attract mff during feeding (DE LEON & DUKE, 1966; SHELLEY et aE., 1979). SHELLEY et al. (1979) reported that S. sanguineurn Kalb ingested 2.3 to 28 times the number of mff present in 1.0 mg of skin. However, these investigators incubated snips for 30 min in distilled water, which would permit 60% or fewer of the mff present to emerge. Our results indicate that S. khruceum ingests-approximately the number of mff in I.0 mg of skin, which could be accounted for by passive intake from the blood pool formed during feeding.

The distribution of mff in our subjects was similar to that found bv DE LEON & DUKE 11966) in their detailed study of four Guatemalans and by SHELLEY et al. (1979) in Brazil. The number of mff was highest on the torso (chest, abdomen and back) and lowest in the legs. With increasing levels of infection, the numbers rose on all sites except the legs, and became high on the shoulder and arms of those with moderate to heavy infections. No site in the torso consistently yielded the highest number of mff.

One purpose of our study was to identify the skin-snip site or sites most suitable for detecting and quantifying onchocercal infections in Guatemala. A single snip taken from the head, shoulder or upper arm correlated well with the over-all level of infection as measured by mean mff intakes by S. ochruceum. However, skin snips from one or two of these sites would have missed infections in two of the five subjects with light infection (Table II). Indeed, subject A was negative in snips taken at all three sites (though a snip previously taken at the shoulder had been positive).

Because concentrations of mff are highest on the torso especially in persons with light or moderate infections, skin snips at these sites are most likely to be positive. However, the density of mff in snips from the lower back was not well correlated with over-all level of infection, since it tended to be high in all subjects. The number of mff ingested by S. ochruceum from all sites except the legs was significantly correlated with the over-all level of infection.

We believe that two or more skin snips from different sites should be taken for quantitative studies of onchocerciasis. A single snip taken from the torso may be suitable, especially when only qualitative information on the prevalence of infection is needed. In this study, the mean of mff emerging from a snip at the head, shoulder or upper arm together with one at the lower back correlated well with the over-all level of infection. In other studies BUNDLING-BENNETT et al. (1981) found the shoulder and the hip to be suitable and easily accessible sites for taking skin snips. Other sites on the torso would be suitable in combination with a skin snip from the head, shoulder or upper arm. Our study does not indicate that the particular combination of sites on the upper body is significantly better than others.

Skin snips from the leg are of little general use in

Page 5: Distribution of microfilariae in Guatemalans with onchocerciasis

258 DISTRIBUTION OF MICROFILARIAE IN GUATEMALANS

Guatemala. Mff may be absent from snips taken trom the legs of lightly infected subjects and densities remain low even in heavily infected subjects.

Investigators have used various measures of skin- snip size to adjust mff counts. Skin-snip weight is most commouly used, but skin-snip area estimated from one or two diameters has also been employed (THOMAS et al., 1973; TADA & FIGUEROA MARRO- QUIN, 1974). We report the results of analysis by skin-snip weight (Tables II and III), because this is the adjustment we have found easiest to make. In our study, adjustment by skin-snip area was equally satisfactory, thbugh no better.

References Armitage, P. (1971). Statistical Methods in Medical Research.

Oxford: Blackwell Scientific Publications, pp. 159-163. Brandling-Bennett, A. D., Anderson, J., Fuglsang, H. &

Collins. R. C. (1981). Onchocerciasis in Guatemala: epide&ology in &cas’ with various intensities of infec- tion. AmekanJournal of Tropical Medicine and Hygiene; 30, 970-981~

Campbell, C. C., Collins, R. C., Houng, A. Y. & Figueroa Marroquin, H. (1980). Quantitative aspects of the infection of Simulium ochraceum by Onchocerca volvulus: the relation of skin micro6larial density to vector infection. Tropenmedizin und Parasitologic, 13, 475-478.

Collins, R. C., Campbell, C. C., Wilton, D. P. & Newton, L. (1977). Quantitative aspects of the infection of Simulium ochraceum by Onchocerca volvulus. Tropenmed- izin und Parasiwlogie, 28, 235-243.

Collins, R. C., Brandling-Bennett, A. D., Holliman, R. B., Campbell, C. C. & Darsie, R. F. (1980). Parasitological diagnosis of onchocerciasis: comparisons of incubation media and incubation times for skin snips. American Journal of Tropical Medicine and Hygiene, 29, 35-41.

Collins, R. C., Merino, M. E. & Cupp, E. W. (1981). Seasonal -trends and diurnal patterns of man-biting activity for four species of Guatemalan Simuliides.

+12c .&wnal of Tropical Medicine and Hygiene, 30,

De Leon, J. R. & Duke, B. 0. L. (1966). Experimental studies on the transmission of Guatemalan and West African strains of Onchocerca volvulus by Simulium ochraceum, S. metallicum and S. callidum. Transactions of the Royal Sociery of Tropical Medicine and Hygiene, 60, 735-752.

Goldman, L. (1944). American onchocerciases. Archives of Dermatology and Syphilis, 50, 385-393.

Omar, M. S. & Garms, R. (1975). The fate and migration of microfilariae of a Guatemalan strain of Onchocerca volvulus in Simulium ochraceum and S. metallicurn and the role of the buccopharyngeal armature in the destruction of microfilariae. Tropenmedizin und Parasitologic, 39, 51-54 __ _ . .

Schulz-Key, H. (1978). A simple technique to assess the total number of Onchocerca volvulus microfilariae in skin snips. Tropenmedizin und Parasitologic, 39, 51-54.

Shelley, A. J., Pinger, R. R., Moraes, M. A. P. & Hayes, J. (1979). Concentration of microiilariae of Onchocerca volvulus by Simulium sanguineurn during feeding: use in mapping parasite distribution in the skin. Journal of Medical Entomology, 16, 48-51.

Tada, I. & Figueroa Marroguin, H. (1974). The density of Onchocerca volvulus microfilariae in the skin at different times of the day in Guatemala. Japanese Journal of Parasitology, 23, 220-225.

Thomas, D. B., Anderson, R. I. 81 MacRae, A. A. (19739 Daytime variation in the density of Onchocerca volvulus microfilariae in human skin. Bulletin of the World Health Organization, 49, 493-498. * I’*

Woodruff, A. W., Choyce, D. P., Muci-Mendoza F., Hills, M. & Petit, L. E. (1966). Onchocerciasis in Guatemala: a clinical and parasitological study with comparisons between the disease there and in East Africa. Traysactions ;{;%pd?“l Socre~ of Troptcal Medtcme and Hygtene, 60,

Accepted for publication 23rd August, 1982.