STANDARD METHOD OF ASSESSING MICROFILARIAL DENSITIES IN ONCHOCERCIASIS 629

CO2 but most died the following day, only one living fluctuations. It is often impossible to time accurately for four days afterwards. the larval development, which is variable from river None of the eggs oviposited developed; they were to river, and which is very important if control all sterile. In this respect it is interesting to note that measures are to be as efficient and economical as no mating was observed in the cage at any time. possible. Artificial breeding-sites of the type des- Lewis et al.f recorded that eggs removed from flies cribed, suitably modified with floats to maintain caught feeding in the field developed in the labor- them at a fixed depth relative to the changing level, atory. This would suggest that the flies mate after would be valuable in this respect. The advantages their first blood meal but may not need to do so after of these breeding-sites are that they are cheap and every blood meal, storing sufficient sperm in their simple to set up, that they require little attention and spermathecae for fertilizing successive batches of that they can be easily examined, either by wading eggs. The spermathecae from the flies caught in the into the river or from a boat, depending on the cage were all empty. depth. Several of them, placed at fixed intervals down a river, would be useful for determining the Discussion effective range of an insecticide, which varies accord- The artificial breeding-site was placed in position ing to the state of the river at any given time. on 5 January 1961 and the cage erected on 19 January, The fact that the cage, apart from providing a but, because of the rapid decrease in the level and useful source of nulliparous flies when used in con- flow of the water, the experiment had to be inter- junction with the sticks, made it possible to induce rupted on 16 February. This note shows that the the flies to bite with relative ease is of paramount methods described were successful in providing in- importance. This paves the way for more research formation on the bionomics of S. damnosum under into the transmission of onchocerciasis. the severe, dry-season conditions prevailing at the This cage was used over a river, but it could easily time, but it is felt that they could be adapted separ- be adapted for keeping flies in the laboratory, where ately and together for more specialized research. conditions can be controlled. By so doing, the opti- While the artificial breeding-site was designed to mum conditions for keeping the flies alive for longer promote more breeding, it could be a useful asset in periods and for inducing feeding could more readily the field ofcontrol, especially in the Savannah district be found. of West Africa. Previous experience in Northern * * Ghana has shown how difficult it is to observe the complete development of any single batch of eggs My thanks are due to Dr G. R. L. Lyons for his helpful through to the pupal stage, especially in the rains, suggestions during the carrying out of the experiment when the rivers are so prone to significant daily described above and for his criticism of the script.

A Standard Method of Assessing Microfilarial Densities on Onchocerciasis Surveys by B. 0. L. DUKE, Senior Medical Research Officer, Helminthiasis Research Unit, West African Councilfor Medical Research, Kumba, Western Cameroon

Assessments of the intensity of infections with aspects of the disease should all be considered in Onchocerca volvulus in human populations, made for conjunction, but the present paper is concerned only research purposes, should include observations on with the estimation of microfilarial densities in the the density of microfilariae in different parts of the body, for it is evident that no two workers adopt the body, on the number and distribution of nodules, same method of assessment and that very few give on the severity and distribution of skin changes, and any details of the methods employed or seem aware on the severity and type of eye lesions. These four of the sources of inaccuracies that confront them. 1193E 630 NOTES

It was shown by Kershaw et al.,a using the method depth from another. Since every operator tends to of weighed skin snips, that the microfilariae of cut slightly differently, it is desirable that the same 0. volvulus conform to definite gross patterns in the person should do all the snipping in any one survey. skin and that repeated snips cut from a given area of skin in the individual at any time of the day or Assessing quantity of skin removed night gave reasonably constant figures for the num- The amount of skin removed in the snip can be bers of microfilariae per milligram of skin in that assessed very much more rapidly and accurately by area. Subsequently, the present author has had con- weighing than by attempting to measure its surface siderable further experience with the method, and area. The weighing is easily done on a torsion while it is true that the density of microfilariae in balance, a model reading up to 10 mg in divisions of adjacent snips is usually approximately constant, yet 0.02 mg being suitable for the purpose. The snip is pocketing of microfilariae may occur so that in some removed from the pin with the aid of a pair of fine cases the assessed densities may vary by as much forceps and is placed on the balance pan with the as 1: 3. The natural irregularities in the minute epidermis downwards. Weighing takes a matter of distribution of microfilariae in a given area of skin seconds, after which the snip is placed on a slide cannot be avoided, but if a reasonably large sample in a drop of normrn saline or in a drop of water of the population is examined such minor errors will ready for teasing. Snips weighing less than 1 mg are cancel each other out. On the other hand, the sites best discarded as they may be too shallow to provide examined on the body, and the assessment of the an adequate sample of the microfilarial population, numbers of microfilariae in the snips taken can be but snips over this weight have a maximum depth done according to a standard and accurate routine, of 300-700 ,u and are deep enough to include all the the xesults of which can be compared directly with layers of the skin in which microfilariae are com- those of other investigators using the same method. monly found. The present paper describes a proven method of assessing microfilarial densities in onchocerciasis and Teasing the snip and time taken for microfilariae to points out some of the sources of error that may be emerge encountered in the fie;d. The rate of emergence of microfilariae from whole Method o taking skin snips skin snips has been found to be so variable that if they are left untouched in saline for 20 minutes the The most satisfactory way of taking a skin snip is proportion of the total contained microfilariae which to use a mounted entomological pin and a safety emerge during that period may be anything from razor blade. The head of the pin is cut off and the 30% to 100%. It is essential therefore that the skin body is mounted in a metal holder in the same way snips should be placed in a drop of saline and then as a bit is held in a brace. The holder is taken in the torn with needles rather than cut into shreds so as left hand and the pin is engaged in the epidermis, to allow all the contained microfilariae to escape. almost horizontally, and lifted so as to raise a cone- This is best done with a pair of Hagedorn needles, shaped fold of skin. The razor blade is held by its held one in each hand as one holds a blackboard two ends between the forefinger and thumb of the chalk, so that the necessary pressure can be exerted. right hand, and the base of the cone is cut through Errors will also arise if the films are counted wet with the blade running parallel to the surface of the too soon after teasing or if they are allowed to dry adjacent skin. A bloodless circular or oval snip before the microfilariae have had time to emerge. about 3-5 mm in diameter and weighing some Serial counts done on freshly teased snips at five- 1-4 mg is thus removed with very little pain, and the minute intervals have shown that immediately after wound is quickly staunched with a small piece of teasing only some two-thirds of the contained micro- cotton wool. It is important always to use pins of filariae have emerged. After five minutes about 90 % the same tensile strength and blades of the same have come out, and constant total counts are ob- degree of suppleness. Without these precautions tained at 10-15 minutes. In order, therefore, to neither the tension that can be exerted to raise the obtain accurate counts the microfilariae must have fold of skin nor the depth of the cut will be constant, time to emerge, and the films must be kept wet for and one series of snips may vary greatly in size and 10-15 minutes before being dried and fixed. In humid tropical climates this presents no difficulty, a Kershaw, W. E., Duke, B. 0. L. & Budden, F. H. (1954) Brit. med. J., 2, 724. but in arid savannah regions some simple humidor STANDARD METHOD OF ASSESSING MICROFILARIAL DENSITIES IN ONCHOCERCIASIS 631

is a necessity in order to prevent the films from in conjunction with the skin snip examination it is drying too fast, and indeed the films may need to be best done by examining each anterior chamber in kept wet by dropping more water on to them during turn through an ophthalmoscope with a high plus the waiting period. lens (a +32 lens is suitable) in the aperture and the light focused right down (i.e., on a Keeler ophthalmo- Examination and preservation of skin snip films scope, the condenser collar should be pulled down The counting and identification of microfilariae as far as it will go). The pupils must first be dilated may be done on wet films, but as counting is a time- with homatropine, and the microfilariae can then consuming process it may preferably be done at be seen as small black silhouettes of characteristic leisure on stained preparations after the return to shape and motility against the light reflected from the laboratory. Great care must be taken to prevent the optic disc and retina. The numbers seen in each microfilariae and shreds of skin from falling off the eye through the pupil aperture can easily be counted. slides during fixation and staining. The use of serum Sites to be examined when assessing microfilarial to stick the films to the slide is unnecessary and densities should be avoided, since it may delay the exodus of microfilariae and it attracts ants. The most satis- In surveying the intensity of infection with factory preservation of films is obtained by simply 0. volvulus in a population large numbers of persons allowing the saline to dry. The drying must be have to be examined. There is not time to take more thorough, and in humid climates, where the salt than two skin snips from each person, nor will the crystals tend to remain hygroscopic, the process subjects normally permit more. It is therefore of must be completed gently over a flame or in an oven. great importance that the two skin snips should be When completely dry the films are fixed in methyl sited so as to reveal as much information as possible. alcohol, and stained in hot Mayer's haemalum. In the course of testing drugs for use against Using this method a correlation of 90% or better 0. volvulus in the forest areas of the Cameroons the between wet and dry microfilarial counts can be intensity of infection in more than 100 persons has obtained. been assessed before treatment b by taking weighed The microfilariae can be counted conveniently at skin snips from the ankles, calves, buttocks, scapulae a magnification of x 50 with a one-inch objective and deltoids on both sides and from the right outer and a x 10 eye-piece on a binocular microscope with canthus, and by counting the microfilariae seen in a movable stage. A hand tally counter is an essential the anterior chamber of each eye. From a considera- aid, the microfilariae being counted thereon in ones, tion of the results obtained it has been possible to fours or tens at the convenience of the operator and decide on the optimum sites for examination in a according to their density in the snip. Once again, routine two-snip survey. At no level was there any it makes for consistency if the same person counts significant difference between the microfilarial den- all the slides obtained on a survey. sities on the right and left sides of the body, although In wet as well as in stained preparations, it will be in early cases unilateral infections may occur. As a found that microfilariae of Dipetalonema streptocerca routine, however, it is advisable always to examine can readily be distinguished from those of 0. volvulus, snips from one side (the right) rather than to in- and their presence does not lead to confusion. vestigate the side that may be thought for various Blood-dwelling microfilariae ofLoa loa or D.perstans reasons to have the greater probability of being are extremely rare in snips that have been taken positive. Of the two sites chosen for skin snips one properly, but when present they can also be re- should be below the waist because, owing to, the cognized easily. biting habits of Simulium damnosum, it is in this It is worth remembering that even after fixation region of the body that early infections are most and staining skin snip films may still be attractive to commonly detected and the highest eventual con- ants and cockroaches and precautions should be centrations of microfilariae develop. For these very taken to prevent these insects from eating the films. reasons the site of election below the waist is the lateral side of the calf. The other of the two sites Assessment of microfilarial densities in the eye should be above the waist; and here the site of This aspect of the investigation may fall within the election is a snip over the deltoid muscle just above of if one province the ophthalmologist is present in b Duke, B. 0. L. (1961) Dissertation for the degree of the survey team, but if the assessment has to be made M.D., Cambridge University. 632 I NOTES its insertion, for the intensity of microfilariae at this eyes and count the numbers of microfilariae seen in site reflects most truly the presence of adult worms the anterior chambers. The fourth man teases the situated above the waist and it is not influenced, as snips and ensures that the slides remain wet for is the scapula snip, by microfilariae, at the'limit of 10-15 minutes before being thoroughly dried and their upward spread, emanating from worms fixed for subsequent transport to the laboratory. If situated below the waist. It is also true that high other aspects of the disease are being investigated counts at the deltoid are more commonly associated simultaneously and other workers are present to with microfilarial invasion of the eye than are high assist, some modification of this routine may, of counts at the scapula. course, be necessary. Finally, the assessment of microfilarial densities in the eyes is made in the manner described above Conclusions by ophthalmoscopic counts of the numbers in the It is considered that the method described in this two anterior chambers. note will give the most accurate assessment attainable of microfilarial densities in populations surveyed for Division of labour in the team assessing microfilarial research purposes. It is one which can be carried densities out successfully under field conditions, and, if To carry out the methods of assessing microfilarial adequate numbers of people from all age-groups are densities described above a trained team of four surveyed in the different topographical environments persons is needed. The first man explains the where onchocerciasis is found, the method will give procedure to the patient, prepares him for examina- the maximum amount of information on the differ- tion, cleans the skin with spirit prior to snipping, ences in microfilarial densities in such populations. staunches the skin wounds and puts mydriatic drops The information so obtained may then be correlated in the patient's eye. The second man, who will with observations made at the same time on eye usually be the medical officer, takes the skin snips lesions, nodule distribution, nutritional status and and passes them on the needle to the third man, genetically determined characteristics. Later it may whose job is to weigh and record. It is also the duty be possible fo correlate microfilarial densities with of the man taking the skin snips to examine the infective biting densities in the Simulium population.

Insecticidal Control of Filariasis * by A. W. A. BROWN, Professor and Head, Department of Zoology, University of Western Ontario, London, Ontario, Canada

The ways in which chemicals are used to control Meanwhile in forested areas in Malaya, Indonesia the mosquito vectors of filariasis are somewhat con- and West Africa, the local Anopheles are capable of ditioned by the species or genus concerned. Vectors acting as vectors of either type of filaria. of Wuchereria malayi in south-east Asia, which are members of the genus Mansonia, complete their Adulticides larval development attached to aquatic weeds such The house-spraying programme with wall deposits as Pistia and Eichhornia. The principal vector of of DDT, so widely used against malaria, has proved W. bancrofti, the tropical house mosquito Culex inadequate to control the vectors of filariasis, even fatigans, develops most strongly in polluted waters when applied at densities of 200 mg per square foot.a close to urban conditions. However, on the Pacific Antimalarial sprays have been reported to be much islands bancroftian filariasis is transmitted by Aides less effective against Culex fatigans than against mosquitos breeding in tree-holes and crab-holes. Anopheles darlingi in British Guiana (Giglioli, 1948),

* Note submitted to the WHO Expert Committee on a A convenient conversion rate is 100 mg per square Filariasis, July 1961. foot = 1 g per square metre. 1193F