A comparison of Mansonella ozzardi microfilaria densities in the blood and in skin snips from three areas of the body

338 TRANSACTIONS OF THE ROYAL SOCIETY OP TROPICAL MEDICINE AND HYGIENE, VOL. 73, No. 3, 1979

A comparison

of Mansonella ozzardi microfilaria densities in the blood and in skin snips from three areas of the body M. B. NATHAN Caribbean Epidemiology

Centre, Port of Spain, Trinidad,

In Brazil, MORAES (1976) and MORAES et al. (1978) reported the presence of Munsonella ozzardi microfilariae in skin snips that were collected using standard onchocerciasis survey techniques. MORAES et al. (1978) and da SILVA et al. (1978) attributed this observation to a tendency of the microfilariae to remain in the capillaries of the dermal papillae, possibly as an adaptation to the biting behaviour of the vector. In Trinidad, West Indies, NATHAN et al. (1978) examined several infected subjects but found relatively few microfilariae in skin snips from six areas of the body when compared with densities in the blood. These low densities in the skin contrast with the Brazilian findings in which high microfilaria densities were often found; it should be noted, however, that smaller biopsies were taken in Trinidad than in Brazil. On the north coast of Trinidad the main vector of M. ozzardi is Culicoides phlebotomus (NATHAN, 1978), a mainly diurnal, exophagous species which feeds predominantly on the legs and ankles. In order to determine whether there is an association between this biting behaviour and the distribution of microfilariae in the skin, as suggested by NELSON & DAVIES (1976), microfilaria densities were compared in biopsies from the shoulder, hip and midcalf regions of infected subjects from this area. Biopsies were taken with a 2 mm Holth punch. To avoid any possible bias due to bilaterally unequal snips were taken microfilaria - concentrations, alternatelv from right and left sides of the bodv in consecutive subjerts. The snips were processed using a modification of the techniques described by SCHEIBER et al. (1976) and BRAUN-MUNZINGER et al. (1977). Thus, unteased snips were placed into separate coded wells of a microtitration plate containing phosphate-buffered saline. The wells were sealed with Scotch tape and left for 24 hours. Well contents were then filtered through 3 pm Nuclepore filters mounted in Swinnex holders. By attaching a canula to the bottom of the holder and a syringe to the top, the well contents could be directlv asuirated on to the filter. Asoiration of the skin snips* was avoided by placing the tip of the canula against the bottom of the well. Each well was rinsed several times with distilled water and the orocedure repeated. A further 10 ml of water was drawn through the assembly to adhere the microfilariae firmlv to the filter. Filters were fixed with methanol, stained for 30 min with Giemsa diluted 1:9 with water buffered at pH 7, gently rinsed with water and dried. The filters were mounted on glass slides under coverslips using immersion oil and

W.I.

microfilariae were identified and counted at x 60 magnification. Skin snips were weighed on a torsion balance and the number of microfilariae per mg of skin calculated. A thick smear was also prepared from 25 mm3 of capillary blood collected with a micropipette from the second finger on the same side of the body that biopsies were taken. Smears were dried for approximaiely 24 hours, stained with Giemsa, and microfilariae were identified and counted at x 60 magnification. Table-Comparison of microfilaria densities of M. ozzardi in skin snips from three sites and from peripheral blood from the, second finger No. mf per 25 mm3 dlood

No. mf per mg skin Shoulder 3.3 1.3 o-0 0.0 ;:; 0.9 0.0 A:; 0.0 2.2 7.1 A:; 0.8 2.4 o-o 1.0 0.0 3.6 8.9 0.0 8.8

Hip 0.0 0.7 o-0 ;:; 0.0 1.0 0.0 2.3 0.5 0.0 1.4 ;:; 6.9 g:; 23.6 12.9 0.0 13.4 3.9 ::;

Calf 0.0 0.0

0 0

;:;

:.

;:;

t

;:;

5

10.7 o-o 0.5 1.1 6.2 o-0 ;:;

1: 12 13 15 24 31 40

:.!: 3.3 o-0 2.0 0.8 6.0 2.3

268 ;z 120 128 141 160

Mean biopsy weights (and ranges) were 2 * 5 mg, (0.6-3.9), 2.9 mg (1.0-4.8) and 2.4 mg (0.5-5.0) for snips from the shoulder, hip and calf respectively. The microfilaria densities are given in the table, with subjects ranked in ascending order of micro-

M.B.NATHAN filaria density in the blood. It can be seen that (a) microfilaria densities in the skin varied widely between the three sites of the body, (b) no one site produced consistently higher microfilaria densities than the others; densities were highest in biopsies from the shoulder in eight subjects, from the hip in six subjects and from the calf in seven subjects, (c) for all three sites the skin densities bore little relation to blood densities. Microfilariae were found in biopsies from two subjects whose thick smears were negative, and in thick smears from two subjects whose biopsies were negative. The highest density was 23 *6 microfilariae per mg of skin in a biopsy from the hip of a male from whom 68 microfilariae were found in 25 mm3 of capillary blood. The inconsistency of the results shows that the variations in microfilaria densities found at the different biopsy sites were not related to the biting behaviour of the vector. The studv bv DUKE (1956) on the intake of Dipetalonema perstans by Cgrahamii and C. inornatipennis from a volunteer who was also infected with Onchocerca volvulus is interesting in that no microfilariae of 0. volvulus were ingested from the volunteer, despite their presence in considerable numbers in the skin at the sites where the Culicoides were feeding. However, Culicoides are also the known vectors of several skin-dwelling microfilariae e.g. D. streptocerca (see CHARDOME & PEEL, 1949; D&x, 1958); Onchocercagibsoni (see BUCKLEY; 1938) and 0. cervicalis (see STEWARD.1933: MELLOR. 1973). Indeed, CHARD&E & PEEL (I949);observing the ingestion of microfilariae by C. grahamii from a carrier of both D. streptocerca and D. perstans, noted that the former were readily picked up from the skin whilst the latter were rarely ingested from the blood. Simulium species are likewise-vectors of both skindwelling microfilariae. e.g. 0. volvulus (see BLACKLOCK, 1926) and 0. gutt&osa (see STEWARD, 1937; EICHLER, 1973), and blood-circulating microfilariae e.g. Ornithoflaria fallisensis (see ANDERSON, 1956). Whilst in the Caribbean, Culicoides species are the vectors of M. ozzardi (see BUCKLEY, 1934; RIPERT et al., 1977; NATHAN, 1978) and Simulium species are reported to be the vectors in South America (CERQUEIRA, 1959; SHELLEY & SHELLEY, 1976). However, the relative importance of the skin and blood-dwelling tendencies of this oarasite in relation to uptake by -the Caribbean and South American vectors requires further elucidation. d

d

\--

-

~I

Acknowledgements This study was funded by the Medical Research Council (U.K.), and was conducted with the support and co-operation of the Trinidad and Tobago Ministry of Health and the Pan American Health Organization (PAHO). I am indebted to Dr. R. Paul, Ministry of Health, for her co-operation and assistance, Mrs. S. Monteil for her technical assistance and to the Director and staff of the PAHO/WHO Caribbean Epidemiology Centre for their advice and encouragement. References Anderson, R. C. (1956). The life cycle and seasonal transmission of Ornithofilaria fallisensis Anderson, a parasite of domestic and wild ducks. Canadian Journal of Zoology, 34, 485.

339

Blacklock, D. B. (1926). The development of Onchocerca volvulus in Simulium damnosum. Annals of Tropical Medicine and Parasitology, 20, l-48. Braun-Munzinger, B. A., Scheiber, P. & Southgate, B. A. (1977). Simplifying modifications to the microtitration plate technique for onchocerciasis surveys. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71, 548-549. Buckley, J. J. C. (1934). On the development, in Culicoides furens Poey, of Filaria ( = Mansonella) ozzardi Manson 1897. Journal of Helminthology, 12, 99-118. Buckley, J. J. C. (1938). On Culicoides as a vector of Onchocerca gibsoni (Cleland & Johnston, 1910). Journal of Helminthology, 16, 121-158. Cerqueira, N. L. (1959). SBbre a transmissao da Mansonella ozzardi. II Nota. Jornal Brazileira de Medicina (Rio), 1, 17-29. Chardome, M. & Peel, E. (1949). La repartition des filaires dans la region de Coquilhatville et la transmission de Dipetolonema streptocerca par Culicoides grahami. Annales de la Socie’te’ Beige de Me’decine Tropicale, 29, 99-119. da Silva, C. J. M., Moraes, M. A. I’. & Cascaes, 0. B. (1978). Mansonella ozzardi: concentra&o das microfilarias nos capilares da pele. Boletim EtGdemioldoico. 10. 97-100. Duke, B. O.-L. (1956). The intake of the microfilariae of Acanthocheilonema perstans by Culicoides grahamii and C. inornatipekzis, and their subseouent develovment. Annals of TroPical Medicine ^ and Parasitology, 50, 32-38. Duke, B. 0. L. (1958). The intake of the microfilariae of Acanthocheilonema streptocerca by Culicoides milnei, with some observations on the potentialities of the fly as a vector. Annals of Tropical Medicine and Parasitology, 52, 123-128. Eichler, D. A. (1973). Studies on Onchocerca gutturosa (Neumann, 1910) and its development in Simulium ornatum (Meigen, 1818). 3. Factors affecting the development of the parasite in its vector. Journal of Helminthology, 47, 73-88. Mellor, P. S. (1973). Studies on Onchocerca cervicalis Railliet and Henry 1910: 1. Onchocerca cervicalis in British horses. Journal of Helminthology, 47, 97-l 10. Moraes, M. A. P. (1976). Mansonella ozzardi in skin snips. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 16. Moraes, M. A. P., Almeida, M. M. R., Lovelace, J. K. & Chaves, G. M. (1978). Mansonella ozzardi entre indios Ticunas do estado do Amazonas, Brasil. Boletin de la Oficina Sanitaria Panamericana, 85, 16-25. Nathan, M. B. (1978). Culicoides phlebotomus, a vector of Mansonella ozzardi in coastal north Trinidad, West Indies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 436-437. Nathan, M. B., Bartholomew, C. F. & Tikasingh, E. S. (1978). The detection of Mansonella ozzardi microfilariae in the skin and blood with a note on the absence of periodicity. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72,420-422. Nelson, G. S. & Davies, J. B. (1976). Observations

340

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ozzardi

h4ICKOFILARIA

DENSITIES

on Mansonella ozzardi in Trinidad. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 16-17. Ripert, C., Raccurt, C. & Douyon, I’. L. (1977). La filariose Munsonella ozzardi en Haiti (Grandes Antilles). Premieres donnees epidemiologiques. Bordeaux Medical, 10, 689-696. Scheiber, I?., Braun-Munzinger, R. A. & Southgate, B. A. (1976). A new technique for determination of microfilarial densities in onchocerciasis. Bulletin of the World Health Organization, 53, 130-133. Shelley, A. J. & Shelley, A. (1976). Further evidence for the transmission of Mansonella ozzardi by

IN BLOOD

AND SKIN

SNIPS

Simulium amazonicum in Brazil. Annals of Tropical Medicine and Parasitology, 70, 212-217: Steward. T. S. (1933). Onchocerca cervicalis (Railliet and Henry 1910) and its development in Culicoides nubeculosus Meigen. Cambridge University Institute of Animal Pathology, 3rd Report, 272. Steward, J. S. (1937). The occurrence of Onchocerca gutturosa Neumann in cattle in England, with an account of its life-history and development in Simulium ornatum Mg. Parasitology, 29,212-213. Accepted for publication

5th January,

1979.

Book Review Primary Child Care. A manual for health workers. Maurice King, Felicity King and Soebagio Martodipoero. Oxford: Oxford University Press, 1978. xi + 315 DD. (illus.). ISBN 0 18 264229 4. Price

E2e.00. - -

.

'

The difficulty in writing text-books for readers whose schooling was limited lies in determining how far to go-in content, extended explanatory detail and simplification of language, while maintaining accuracy, relevance and reader-interest. Omitted ‘ essentials ’ provoke criticism while language simplification invites circumlocution. Jargon&e is, after all, trade shorthand. The subiect of Primarv Child Care is an ambitious one including, as it does; not only the services which a child in a developed country might obtain outside the hospital but also many of the procedures which, in the case of the developing countries, must take mace in the home, in the village primary health centre or, at best,. in the extended health centre which has some in-oatient facilities. The authors address their work to anybody who can read it and, understand words like organism, presumably, dehvdration and svmotomatic. The level of semantic sophistication varied from chapter to chapter. Molluscum contagiosum and gonococcal vulvovaginitis get complete billing but the chapter on tuberculous diseases never spells out the noun but repeatedly chirps ‘TB’. This book is not an especially innovative teaching tool. Reliance on reading skills will necessarily exclude the increasingly important body of village or intermediate level health workers. It strongly resembles the manuals, written-down medical textbooks, long developed for home, workshop or battlefield to permit the untrained to deal with medical emergencies in the absence of conventional medical personnel. It does not attempt to lead the reader through any novel approach to treating sickness through modules of observation and examination without necessarily putting a name to what is then treated. New programmes like Medex have found such training by modules more useful than attempting to create scaled down physicians. The book’s disease-oriented chapters begin with symptomatology-or with the area of the body of concem-

and then provide a conventional review of the anatomy and pathology of disease before getting down to the more practical issues of the signs of disease and how to observe, manage and treat the patient. The actual steps of examination are made clear by some good simple drawings. Especially helpful are the suggestions on what to tell the mother of the sick child. Instructions in small black type stand out from the general text and so enable the book to be used as a manual when the health worker is confronted by a patient. The work is consequently a compromise between an explanatory text on the causes and progress of disease and a practical manual on how to deal with specific complaints. There are some odd editorial decisions. For examole. in the section on tuberculosis (TB) the aetiology of the disease is well summarized’ and points such as INH resistance in adults and the diagnostic use of X-rays in children are described. There is, however, no mention of the use of tuberculin testing in child case-contacts or of INH resistant BCG for the infants of nursing mothers under treatment for the disease. The book is probably a little long, as well as academic, for workers, even if they are skilled readers, who are less privileged in their professional training. The final drug treatment guide is clear once the symbols have been mastered. The softback book is well printed, sturdy and at two pounds sterling very cheap. For the medical student or high technology accustomed physician this is a beautiful ‘back to basics’ publication. Whether or not all the advice, such as-on the keeping of ‘special risk’ registers of babies to be followed up by home visits, originating as it does in the affluent western society, is appropriate for many developing countries at least it measures shortcomings with the ideal. It will likely prove most attractive ;o the already trained nurse who wishes to extend her competence to provide unsupervised primary care. Evidently it was not designed for the primary health care workers found in most developing countries and this limits its usefulness. However, it may prove very useful for their teachers. DEREK ROBINSON