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HUMAN ONCHOCERCIASIS: CRYOPRESERVATION OF ISOLATED MICROFILARIAE
450 correlation (r values): 0 - 02 (fluorophotometry), 0 . 25 (oscillatory
potential),
0 - 09
(macula
time), 0-32 (glomerular filtration rate), and 0 - 08 (albuminuria); all are non-significant (n=31 for all correlations). Thus, in sporting terms, the question raised by Hockaday is "off side". The reproducibility (coefficient of variation) for the measures of eye function, when taking the mean of two eyes for each patient, is less than 5%. Hockaday asks about the arbitrary units for macula recovery time. We used the stepwise increase in visual acuity (0 - 1, 0 - 2, and so on) as a function of time during the initial 2 min period of macular readaptation following a similar period of exposure to very bright light. For each step of visual acuity the area under the curve was determined and the separate values were finally added. I apologise for the conflict between text and table 11 that Hockaday notes: the second largest urinary albumin excretion rate in the total population (n 31) is 840 g/min; in the CSII group it is 320 tg/min. The randomisation was, as Hockaday notes, between two recovery
=
different methods of injection and the results were, in this interim report, given for the two treatment groups. However, since we too believe that it is the level of blood glucose control rather than the manner of injection that matters, further reports will be on patients divided into groups according to different levels of blood glucose control. Steno Memorial Hospital, DK-2820 Gentofte, Copenhagen, Denmark
TORSTEN
LAURITZEN,
On behalf of the Steno
Study Group
BLEEDING TIMES IN SOUTHERN SWEDEN
SIR,-Dr Gustafson and I agree that differences in bleeding times between our study (Nov. 28, p. 1190) and others, pointed out by Dr
Dyerberg (Feb. 13, We have done
p.
more
398) require comment. bleeding time determinations on apparently
healthy volunteers, all non-smokers and in the same age group. The table compares our results with those obtained by the same observer
HUMAN ONCHOCERCIASIS: CRYOPRESERVATION OF ISOLATED MICROFILARIAE
SIR,-The filarial nematode Onchocerca volvulus is the cause of African and Central and South American river blindness. Research has been hampered by the lack of readily available parasitic material of human origin. Cryopreservation would be a useful tool for the transport of living material to countries with research facilities. However, the desirable state for freezing the skin-dwelling microfilariae would be free of host tissue, and this has never been achieved with 0. volvulus. Microfilariae have been frozen in skin biopsy specimens but no measure of efficiency could be made. Such techniques result in the loss of a large proportion of damaged parasites which fail to migrate out of the tissues.2 Recent studies have led to the development of an efficient technique for cryopreserving 0. lienalis and 0. cervicalis microfilariae (bovine and equine dwelling parasites, respectively) free of their host tissue, in liquid nitrogen, resulting in about 78% infectivity on thawing. We have now applied this technique to human parasites collected at an endemic focus of onchocerciasis, the town of Wau in the Bahr El Ghazal region of southern Sudan. Adult worms were removed from thirty-five patients by nodulectomy and the skin overlying these subcutaneous nodules was also removed. Sterile slivers of this skin (usually from the iliac crest region) were incubated in sterile 199 medium (Wellcome Reagents Ltd) for 2 h at 28° C and the emerging microfilariae were cryopreserved by the technique described by Ham et al. This involved a two-stage incubation of the parasites in solutions of ethanediol, after which they were simply dropped into liquid nitrogen. The advantage of this technique is its suitability to field conditions; no complex equipment is required, only some ice and some liquid nitrogen. At -196°C there appears to be no deterioration of viability with time, and examination of some thawed microfilariae showed that about 95% appeared normally motile, a figure similar to that obtained with the bovine and equine
species.
BLEEDING TIMES IN YOUNG MALE VOLUNTEERS
Around 300 000 microfilariae were collected, cryopreserved, and flown to Britain where the material is required for work on immunology, chemotherapy, vector susceptibility, and in vitro
development. This work was supported by Programme in Tropical Diseases. Difference between (A) and (B) not significant; between (A) and (C) and between (B) and
(C)
p
Eye Hospital,
Department of Medical Helminthology,
British volunteers, also non-smokers and in the same age group; with those of Nilsson et a1. on other Swedes, whose age range and smoking habits were not indicated; and with bleeding times determined on British volunteers by Treacher et a1.2 The bleeding time of healthy young males is significantly longer in southern Sweden than it is in London and, according to Dyerberg, in Denmark also. Now that the validity of the reported differences has been confirmed, possible reasons can be considered. Technical differences seem an unlikely explanation for the disparity between Lund and London, because the determinations were made by the 2 same observer and the results accord with those reported by others. Presumably, therefore, the results indicate differences in one or more components of primary haemostasis. It would be interesting if these differences were due to dietary habits. Whatever the explanation, there is evidence that a diet similarly enriched with eicosapentaenoic acid causes the bleeding time, whether shorter or longer initially, to be increased in the same proportion (i.e., by about 40%). Department of Internal Medicine, University Hospital of Lund,
London School of Hygiene and London WC1E 7HT
Tropical Medicine,
P. J. HAM
POPCORN AND FAIRIES IN THE MANAGEMENT OF MEASLES IN ETHIOPIA
SIR,-We may be able to improve our teaching of home care in the management of certain diseases if we understand local beliefs about causation and local customs in care. Amongst the children of the 4 Third World severe measles is the rule rather than the exception.4 Hospital care is rarely available and measles immunisation continues to be virtually absent in many countries, so ways of managing the disease at home are still needed. Thirty-four refugee mothers from Tigray in northern Ethiopia, at the Um Gulja camp in eastern Sudan were asked during July and August, 1981, what is done when children have measles. Several Tigrigna names are used for measles (fremai, nefyo, godif, wenesh).
MARGARET THORNGREN 1. Schiller
1. Nilsson
L, Tengborn L, Nilsson SM. Ett nytt hjäpmedel for standardisering av blödningstidsbestämning. Opuscula Med 1979; 24: 65-66 2. Treacher D, Warlow C, McPherson K. Aspirin and bleeding-time. Lancet 1978, ii: 1378. 3. Sanders
H. EL SHEIKH
Khartoum, Sudan
on
S-221 85 Lund, Sweden
Khartoum
the U.N.D.P./World Bank/W.H.O.
TAB, Vickers M, Haines AP. Effect on blood lipids and haemostasis of supplement of cod-liver oil, rich in eicosapentaenoic and docosahexaenoic acids, in healthy young men. Clin Sci 1981, 61: 317-24.
vitro
EL, Turner VM, Marroqúin HF, D’antonio R. The cryopreservation and m cultivation of larval Onchocerca volvulus. Am J Trop Med Hyg 1979; 28:
997-1009. 2. Ham PJ, Bianco AE. Quantification ofa cryopreservation technique for Onchocerca microfilariae in skin snips. J Helminthol 1981; 55: 59-61. 3. Ham PJ, Townson S, James ER, Bianco AE. An improved technique for the cryopreservation of Onchocerca microfilariae. Parasitol 1981; 83: 139-46. 4. Morley DC. Measles in developing countries. Proc Roy Soc Med 1974; 67: 1112.
potential),
0 - 09
(macula
time), 0-32 (glomerular filtration rate), and 0 - 08 (albuminuria); all are non-significant (n=31 for all correlations). Thus, in sporting terms, the question raised by Hockaday is "off side". The reproducibility (coefficient of variation) for the measures of eye function, when taking the mean of two eyes for each patient, is less than 5%. Hockaday asks about the arbitrary units for macula recovery time. We used the stepwise increase in visual acuity (0 - 1, 0 - 2, and so on) as a function of time during the initial 2 min period of macular readaptation following a similar period of exposure to very bright light. For each step of visual acuity the area under the curve was determined and the separate values were finally added. I apologise for the conflict between text and table 11 that Hockaday notes: the second largest urinary albumin excretion rate in the total population (n 31) is 840 g/min; in the CSII group it is 320 tg/min. The randomisation was, as Hockaday notes, between two recovery
=
different methods of injection and the results were, in this interim report, given for the two treatment groups. However, since we too believe that it is the level of blood glucose control rather than the manner of injection that matters, further reports will be on patients divided into groups according to different levels of blood glucose control. Steno Memorial Hospital, DK-2820 Gentofte, Copenhagen, Denmark
TORSTEN
LAURITZEN,
On behalf of the Steno
Study Group
BLEEDING TIMES IN SOUTHERN SWEDEN
SIR,-Dr Gustafson and I agree that differences in bleeding times between our study (Nov. 28, p. 1190) and others, pointed out by Dr
Dyerberg (Feb. 13, We have done
p.
more
398) require comment. bleeding time determinations on apparently
healthy volunteers, all non-smokers and in the same age group. The table compares our results with those obtained by the same observer
HUMAN ONCHOCERCIASIS: CRYOPRESERVATION OF ISOLATED MICROFILARIAE
SIR,-The filarial nematode Onchocerca volvulus is the cause of African and Central and South American river blindness. Research has been hampered by the lack of readily available parasitic material of human origin. Cryopreservation would be a useful tool for the transport of living material to countries with research facilities. However, the desirable state for freezing the skin-dwelling microfilariae would be free of host tissue, and this has never been achieved with 0. volvulus. Microfilariae have been frozen in skin biopsy specimens but no measure of efficiency could be made. Such techniques result in the loss of a large proportion of damaged parasites which fail to migrate out of the tissues.2 Recent studies have led to the development of an efficient technique for cryopreserving 0. lienalis and 0. cervicalis microfilariae (bovine and equine dwelling parasites, respectively) free of their host tissue, in liquid nitrogen, resulting in about 78% infectivity on thawing. We have now applied this technique to human parasites collected at an endemic focus of onchocerciasis, the town of Wau in the Bahr El Ghazal region of southern Sudan. Adult worms were removed from thirty-five patients by nodulectomy and the skin overlying these subcutaneous nodules was also removed. Sterile slivers of this skin (usually from the iliac crest region) were incubated in sterile 199 medium (Wellcome Reagents Ltd) for 2 h at 28° C and the emerging microfilariae were cryopreserved by the technique described by Ham et al. This involved a two-stage incubation of the parasites in solutions of ethanediol, after which they were simply dropped into liquid nitrogen. The advantage of this technique is its suitability to field conditions; no complex equipment is required, only some ice and some liquid nitrogen. At -196°C there appears to be no deterioration of viability with time, and examination of some thawed microfilariae showed that about 95% appeared normally motile, a figure similar to that obtained with the bovine and equine
species.
BLEEDING TIMES IN YOUNG MALE VOLUNTEERS
Around 300 000 microfilariae were collected, cryopreserved, and flown to Britain where the material is required for work on immunology, chemotherapy, vector susceptibility, and in vitro
development. This work was supported by Programme in Tropical Diseases. Difference between (A) and (B) not significant; between (A) and (C) and between (B) and
(C)
p
Eye Hospital,
Department of Medical Helminthology,
British volunteers, also non-smokers and in the same age group; with those of Nilsson et a1. on other Swedes, whose age range and smoking habits were not indicated; and with bleeding times determined on British volunteers by Treacher et a1.2 The bleeding time of healthy young males is significantly longer in southern Sweden than it is in London and, according to Dyerberg, in Denmark also. Now that the validity of the reported differences has been confirmed, possible reasons can be considered. Technical differences seem an unlikely explanation for the disparity between Lund and London, because the determinations were made by the 2 same observer and the results accord with those reported by others. Presumably, therefore, the results indicate differences in one or more components of primary haemostasis. It would be interesting if these differences were due to dietary habits. Whatever the explanation, there is evidence that a diet similarly enriched with eicosapentaenoic acid causes the bleeding time, whether shorter or longer initially, to be increased in the same proportion (i.e., by about 40%). Department of Internal Medicine, University Hospital of Lund,
London School of Hygiene and London WC1E 7HT
Tropical Medicine,
P. J. HAM
POPCORN AND FAIRIES IN THE MANAGEMENT OF MEASLES IN ETHIOPIA
SIR,-We may be able to improve our teaching of home care in the management of certain diseases if we understand local beliefs about causation and local customs in care. Amongst the children of the 4 Third World severe measles is the rule rather than the exception.4 Hospital care is rarely available and measles immunisation continues to be virtually absent in many countries, so ways of managing the disease at home are still needed. Thirty-four refugee mothers from Tigray in northern Ethiopia, at the Um Gulja camp in eastern Sudan were asked during July and August, 1981, what is done when children have measles. Several Tigrigna names are used for measles (fremai, nefyo, godif, wenesh).
MARGARET THORNGREN 1. Schiller
1. Nilsson
L, Tengborn L, Nilsson SM. Ett nytt hjäpmedel for standardisering av blödningstidsbestämning. Opuscula Med 1979; 24: 65-66 2. Treacher D, Warlow C, McPherson K. Aspirin and bleeding-time. Lancet 1978, ii: 1378. 3. Sanders
H. EL SHEIKH
Khartoum, Sudan
on
S-221 85 Lund, Sweden
Khartoum
the U.N.D.P./World Bank/W.H.O.
TAB, Vickers M, Haines AP. Effect on blood lipids and haemostasis of supplement of cod-liver oil, rich in eicosapentaenoic and docosahexaenoic acids, in healthy young men. Clin Sci 1981, 61: 317-24.
vitro
EL, Turner VM, Marroqúin HF, D’antonio R. The cryopreservation and m cultivation of larval Onchocerca volvulus. Am J Trop Med Hyg 1979; 28:
997-1009. 2. Ham PJ, Bianco AE. Quantification ofa cryopreservation technique for Onchocerca microfilariae in skin snips. J Helminthol 1981; 55: 59-61. 3. Ham PJ, Townson S, James ER, Bianco AE. An improved technique for the cryopreservation of Onchocerca microfilariae. Parasitol 1981; 83: 139-46. 4. Morley DC. Measles in developing countries. Proc Roy Soc Med 1974; 67: 1112.