A new technique for the determination of microfilarial densities in onchocerciasis*

BRIEF COMMUNICATIONS A new technique for the determination of microfilarial densities in onchocerciasis * P. SCHEIBER,1 R. A. BRAUN-MUNZINGER,' & B. A. SOUTHGATE 2 Abstract Precise measurement of the parasite load in helminthic infections makes it possible to correlate morbidity, the individual response to treatment, and the best control methods. A membrane filtration technique was recently developed for quantifying Wuchereria bancrofti microfilaraemia. The present paper describes its application to onchocerciasis in a study on 107 patients in northern Togo. The technique is cheap and simple to apply under field conditions, and is extremely efficient at recovering microfilariae from skin snips. The importance of precise measurement of the parasite load carried by individuals and communities in helminthic infections has been recognized for many years. Parasite loads may determine the amount of disease produced, the response to treatment of the individual patient, and the choice of methods to be used for control of the infection in the community. The accurate assessment of the density or output of the particular stage in the parasite life- cycle that is responsible for the continuance of transmission among human hosts is essential, espe- cially: (a) for measuring the intensity of transmission and infection in a community; (b) as a guide to the development of pathological conditions in the community and, hence, for deter- mining the public health importance of the infection and the priority of its control; (c) for providing data needed in predictive, quan- titative, epidemiological models of transmission, upon which a rational choice of control measures can be based; and * This study was supported by the Ministry of Public Health and Social Affairs, Republic of Togo, and by the German Agency for Technical Cooperation (GTZ), Esch- born, Federal Republic of Germany. 1 The Ernst Rodenwaldt National Institute of Hygiene, Lome, Togo. " Ross Institute of Tropical Hygiene, London School of Hygiene and Tropical Medicine, London, England. Requests for reprints should be addressed to Dr B. A. Southgate. (d) for providing baseline data for the evaluation of such control measures as are put into effect. In the case of Onchocerca volvulus infections, such measurements have hitherto depended upon count- ing the microfilariae emerging from skin snips. These counts are performed in the field shortly after the snip has been taken. The most commonly used tech- nique, described by Kershaw et al. (15) and by Duke (11), consists in taking a conical-shaped skin snip with a mounted pin and razor blade, teasing it rapidly in isotonic saline on a glass slide, and count- ing the microfilariae that emerge in 10-15 minutes, by means of a microscope at a magnification of x 25 or x 50. This method has been used successfully in a number of onchocerciasis studies (1, 12-14). The relative merits of the basic technique and of various recently introduced modifications (16, 17) have been reviewed by Buck (6), and further improvements in field quantification of microfilarial densities in onchocerciasis have been described by Brinkmann (3, 4), who devised methods for obviating the use of analytical balances in field work. However, all the techniques in use at the present time suffer from the following disadvantages: (a) counting must be performed relatively soon after the skin snip has been taken, often before all microfilariae have emerged; (b) as a result of this, slight infections may be missed and the true prevalence underestimated; (c) in heavy infections, the hundreds of microfila- riae that emerge from a single skin snip cannot be counted accurately, and repeated counts are impos- sible owing to drying of the preparations; (d) counting and skin snipping must proceed simultaneously, thereby increasing the number of personnel required in the field team and prolonging the work of taking skin snips so far into the day that variations in results may be produced owing to diurnal fluctuations in microfilarial density; (e) inexperienced workers may find it very difficult to distinguish the motile, unstained microfilariae of 3425 - 130 BULL. WORLD HEALTH ORGAN., Vol. 53,1976 BRIEF COMMUNICATIONS 131 0. volvulus from those of Dipetalonema streptocerca and occasional diurnally present blood-borne micro- filariae, such as those of D. perstans and Loa loa; and (f) in our experience the type of worker best suited to the accurate identification and counting of parasites is often unable to perform as efficiently under difficult and trying field conditions. The importance of determining microfilarial densi- ties, and the imperfections of the techniques cur- rently used, have led us to develop a new technique for the enumeration of microfilarial densities in onchocerciasis surveys, based on the use of mem- brane-filtration with Millipore filters a and Swinnex filter holders.a This technique was recently developed for the quantification of Wuchereria bancrofti microfilar- aemia (2, 7). In subsequent studies the use and ease of application of the technique in field conditions were described (8-10). Its capacity to detect ultra-low- density microfilaraemia and the potential epidemi- ological importance of this feature were stressed (5) and the radical differences that it revealed in the epidemiological pattern in a community were clearly indicated (18). This paper describes the adaptation of the mem- brane-filtration technique to onchocerciasis, gives some preliminary results, and indicates some advan- tages of the new method. Materials and methods Location of study area. The study was carried out in a savanna focus of onchocerciasis surrounding Ogaro village in the region of Dapango, northern Togo, during March 1975. All persons examined belonged to the Gourma tribe. Selection of subjects for examination. As the study was concerned only with the development of a technique, no attempt at random sampling was made. Skin snips were taken only from persons exhibiting typical onchocercal nodules, regardless of age and sex. Technique ofskin snips. Skin snipping was regular- ly performed between 10.00 and 14.00 hours, to minimize variations in microfilarial densities during the day. All snips were taken from the upper lateral quadrant of the right buttock below the iliac crest, with a sterile Walser-type corneoscleral punch.b As it a Manufactured by Millipore Corporation, Bedford, MA, USA. b Manufactured by Leonhard Klein, Heidelberg, Federal Republic of Germany. was not necessary for the purposes of this study, no attempt was made to measure the surface area or determine the weight of the skin snips. Immediately after they were taken, the specimens were placed in a well of a 0.3-ml polystyrene microtitration plate that had been filled previously with either isotonic saline or distilled water. Both " V "-shaped and " U "- shaped plates were used, but the latter were found to be more satisfactory. The letter and number codes of the appropriate well were recorded on the patient's history and clinical examination form. Before the plates were transported from the place of skin snipping to the field laboratory, each plate was covered with a sheet of Filmoplast c to prevent evaporation, the entry of dust, and spilling of the well contents. Filtration of microfilariae. Several hours later, in the field laboratory, the sheet of Filmoplast was removed and the wells were rapidly scanned under a binocular microscope at x 25 magnification to determine the presence of microfilariae; although many snips were apparently negative at that time, all of them subsequently showed microfilariae after filtration. Each snip was then carefully teased with needles, the microtitration plate was covered again with Filmoplast, and the preparations were left for 1 h. At the end of that time, the plates were uncovered and the skin snip was transferred to an unused, coded well filled with isotonic saline; the piece of skin and the needle carrying it were washed with saline into the original well before the transfer. The saline from each original well was then aspirated into a 2-ml syringe, the plate being lifted slowly into the vertical position during the process. The contents of the well were then gently passed through a 25-mm diameter Millipore filter of 5-,tm pore size held in a Swinnex filter holder. The syringes were refilled with clean saline, which also passed through the filter. Then 3 syringes-full of air were passed through the filter to ensure firm adhesion of the microfilariae. After the completion of this filtration process, the empty wells were again scanned with a microscope; wells into which skin snips had been transferred were examined microscopically 6-12 h later. Staining of filters. Filters were dried carefully, stored in a dry box, and transported back to the central laboratory in Lome at the end of the field study. They were stained for 1 h in Giemsa stain c Manufactured by Haus Neschen (Filmoplast), Buecke- burg, Federal Republic of Germany. 132 BRIEF COMMUNICATIONS diluted 1: 10 in phosphate-buffered water at pH 7.2 and subsequently differentiated for 1 min at pH 7.2. Mounting and examination of filters. After stain- ing, the filters were dried overnight in an incubator and mounted on a glass slide under a coverslip in Caedax.a They were examined microscopically at a magnification of x 60 and all microfilariae present were identified and counted. Results Table 1 shows the age and sex distribution of the 107 subjects examined. Table 2 gives detailed data on the counting of microfilariae. All the persons examined were positive and the 107 skin snips yielded a total of 19 333 microfilariae. Microscopic scanning of wells immediately after removing the fluid for the filtration process revealed no remaining parasites. Table 1. Age and sex distribution of the subjects examined Age in years Males Females Total 5-14 0 1 1 15-44 44 52 96 45+ 8 2 10 Total 52 55 107 Table 2. Millipore filter counts of microfilariae in 107 skin snips from males and females Males Females range of counts 19-803 8-559 mean count 204 159 median count 158 119 Out of the 76 wells with transferred skin snips that were examined 6-12 h after filtration, 29 were posi- tive for microfilariae and 47 negative. The range of microfilarial counts in positive wells was 1-8, the mean count 2.4, and the median count 2. The mean count in all 76 wells examined was 0.9. The total number of microfilariae counted in the positive wells was 69-i.e., 0.36%4 of all those that emerged. a Manufactured by E. Merck, Darmstadt, Federal Republic of Germany. After staining, the morphology of 0. volvulus microfilariae was clearly revealed and precise species identification presented no problems. Discussion From the results indicated above, it can be seen that the new technique is extremely efficient, since it enabled us to recover virtually all microfilariae capable of emerging from skin snips, whereas only 0.36°/ emerged after the filtration process. The method is cheap, quick, and simple to apply under field conditions, and it can be performed successfully without the presence of highly skilled and experienced workers. All six major disadvan- tages of the present standard techniques (see page 130) are avoided. It is proposed to carry out a full- scale epidemiological study in the near future, using the standard and new techniques simultaneously on the same population. ACKNOWLEDGEMENTS We are grateful for the technical assistance of Mr Leo- nard Lawson. REFERENCES 1. ANDERSON, J. ET AL. The prognostic value of head nodules and microfflariae in the skin in relation to ocular onchocerciasis. Tropenmedizin und Parasito- logie, 26: 191-195 (1975). 2. BELL, D. 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