Biomedical subjects
B O Duke
Publications and source records attributed to B O Duke.
Effects of three-month doses of ivermectin on adult Onchocerca volvulus.
Onchocerca volvulus worms in nodules from Guatemalan patients treated with four, eight, or 11 single doses of ivermectin (150 micrograms/kg of body weight) that were given once every three months were examined by routine histologic techniques and compared with worms in control nodules from untreated persons living in the same location over the same time periods. All treated nodules were removed four months after the last dose of ivermectin, i.e., 13, 25, or 34 months after the start of the trial. At the 25th and 34th months, i.e., after the eighth or eleventh doses of ivermectin, there were excess mortalities in female worms of 25.5% and 32.6%, respectively, over and above the levels in controls. Furthermore, the proportions of live females still producing scanty embryos up to the gastrula stage were only 7.7% and 18.2%, and no females were producing microfilariae. Ivermectin given at 3-month intervals also reduced significantly the mean numbers of live male worms in nodules, as well as the proportions of inseminated females. This regimen was effective in preventing embryogenesis to the microfilarial stage while, at the same time, it caused a slow but steady attrition of the adult worms.
The selection of communities for treatment of onchocerciasis with ivermectin.
In areas endemic for onchocerciasis, active community-based treatment with ivermectin is preferred to individual diagnosis and treatment. Ideally, all infected persons should be treated, although initially priorities may have to be set at local or national levels. We suggest that all communities with a prevalence of O. volvulus infection of 20% or more in adult males aged 20 years or over should be treated; and that elsewhere facilities for passive treatment should be provided. In some areas, for logistical reasons, treatment may first have to be started in communities with the highest prevalences (perhaps above 40% or even 60%) and then expanded to all endemic communities. The available data suggest that a rapid assessment method based on the examination for nodules will give a simple, acceptable, non-invasive and reasonably reliable method of identifying the communities that should be treated. If a nodule is detected in at least three men from a sample of 30 men aged 20 years or over, the community can be assumed to have a true prevalence of infection of 20% or more and should be included in community-based treatment.
Observations and reflections on the immature stages of Onchocerca volvulus in the human host.
Knowledge of the morphology, migrations and development rates of the L3, L4 and prepubertal L5 stages of Onchocerca volvulus in the vertebrate host is an essential prerequisite to work vaccine development of chemoprophylaxis. After a brief review of the scanty literature available on this subject a description is given of two immature worms, one male and one female, found in an O. volvulus nodule from Guatemala. The male, which measured 12 mm x 40 micron(s), had fine regular transverse cuticular annulations, two prominent spicules, and an undifferentiated testis. The female, which measured 20 mm x 40 micron(s), had not yet developed cuticular ridges but showed a vulva, gut, and two rudimentary genital tracts. It is thought that these worms may have been six to 12 weeks old. The likely course of the early development of O. volvulus in the vertebrate host is discussed, based on analogy with O. lienalis in cattle; and some of the factors to be taken into account when pursuing the search for O. volvulus immatures are described.
Comparison of the effects of a single dose and of four six-monthly doses of ivermectin on adult Onchocerca volvulus.
Adult Onchocerca volvulus worms, extracted from nodules of Guatemalans by collagenase digestion, were examined whole and by histological techniques. One group of persons received a single 150 micrograms/kg dose of ivermectin; two other groups (one with older and one with younger nodules) received four similar doses of ivermectin at 6-month intervals. For each group, there were comparable untreated controls. All nodules were removed six months after the last dose. After a single dose, the only significant difference from the controls was in the decreased proportion of female worms producing live microfilariae. After four doses, there were significant increases in the proportions of moribund/dead female worms and of live uninseminated females, when compared with the corresponding controls. There were also fewer male worms present, but this difference was not significant. Six months after the conclusion of the 4-dose regimen, the proportion of female worms producing live microfilariae was significantly lower than in the groups that had received a single dose.
Suramin and the time it takes to kill Onchocerca volvulus.
The paper records the numbers of 1-2 mm shotty papules developing in the author's skin after 2-day courses of diethylcarbamazine (DEC) repeated every 16 days before, during and after a 7.1 G suramin course for cutaneous onchocerciasis. Assuming, from biopsy evidence, that each papule represented a dead Onchocerca volvulus microfilaria (mf), the number of mfs reaching the skin every 16 days did not begin to fall until 96 days after the first full dose of suramin; and only reached zero by day 224. The histopathology of nodules excised from Cameroonian patients before, and at intervals of 56-335 days after, the start of a 7.1 G suramin course, revealed changes in the worms that correlated over time with the disappearance of mfs from the skin. Suramin sterilized and killed the male worms between days 77 and 105 and, in the females, it adversely affected the staining and subsequent development of small morulae from about day 56. It was estimated that new embryogenesis ceased about 56-77 days after the first full dose; development of the last viable embryos to mfs was complete by about 136 days; and the last mfs, perhaps having reduced vitality, emerged from the females by 160 days and reached the skin within 16-32 days.
The embryogenesis of Onchocerca volvulus over the first year after a single dose of ivermectin.
Adult Onchocerca volvulus, extracted from nodules before, and at intervals of two weeks to 12 months after, a single 150 micrograms/kg dose of ivermectin, were examined longitudinally and by sequential transverse sections. The mean number of male worms per nodule fell, and the proportion of nodules with no male worm rose, within two weeks of ivermectin and remained so for 12 months. In female worms, at intervals after ivermectin, the percentages of the length of the lower genital tracts occupied by embryos at each stage of development, or by degenerating ova, embryos and microfilariae (mfs), were recorded: (a) in un(re-)inseminated worms whose original embryogenesis was continuing and in those in which it was completed; and (b) in worms, reinseminated post-ivermectin, in which a new embryogenesis had begun. The results indicated that: (a) the time needed for the zygotes of O. volvulus to develop to mfs is 8-12 weeks; (b) nearly 40 percent of females had not resumed mf production by 12 months after treatment; (c) many intrauterine mfs had not degenerated within the first two weeks of ivermectin; (d) some of the last embryos to mature to mfs did not degenerate but accumulated temporarily in the anterior uteri 8-16 weeks after ivermectin.
Viability of adult Onchocerca volvulus after six 2-weekly doses of ivermectin.
Ivermectin is a safe, effective microfilaricide and microfilarial suppressant for Onchocerca volvulus; but in single doses of 100-200 micrograms/kg body weight it has no macrofilaricidal action. The present trial aimed to determine whether 6 doses of 100 micrograms/kg ivermectin, given at 2-week intervals, would kill the adult worms. Eighty-two nodules from 28 otherwise healthy adult male Liberian patients treated with this ivermectin schedule, and 102 nodules from a similar group of 25 control patients, were removed four months after the last dose of ivermectin. They were coded and assessed in a masked fashion either by routine histology or by examination of whole worms extracted from the nodules after collagenase digestion. The drug had no visible effect on adult male worms. More adult female worms were assessed as moribund or dead in the ivermectin-treated group than in the control group (for the collagenase digests P = 0.09; for the histological assessment P = 0.47). The data suggest that repeated dosage with ivermectin may lead to a slow attrition of some female worms and this possibility should be investigated in patients receiving regular doses every 3, 6 or 12 months as part of onchocerciasis control programmes.
Migration and death of skin-dwelling Onchocerca volvulus microfilariae after treatment with ivermectin.
The effects of a single dose of ivermectin (122-200 micrograms/kg) on Onchocerca volvulus microfilariae (mf) in skin and lymph nodes were studied histologically in a qualitative and quantitative manner over seven days after treatment. Ivermectin caused mf to move from the subepidermal layer into the deeper layers of the dermis, subcutaneous fat and connective tissue, and the regional lymph nodes. No mf were seen migrating upwards through the epidermis. Dead mf were not seen before 24 hr and the numbers found dead in the dermis were far fewer than the total numbers in the skin before treatment. Inflammatory cellular reaction around dead mf in the tissues was usually minimal. The results suggest that most of the mf which disappear from the skin may be destroyed in the lymph nodes and that the histological reaction excited by mf dying under ivermectin is less violent than that after diethylcarbamazine.
Effect of diethylcarbamazine on adult Loa in monkeys.
The effect of diethylcarbamazine citrate (DEC) treatment (43-150 mg kg-1 daily for 12-21 days) on adult Loa worms in five drills (Mandrillus leucophaeus) is described. In individual monkeys, between one-third and all of the adult worms survived the treatment, and most of the surviving females were still producing live microfilariae. These experiments provide direct evidence which may explain the common clinical experience that single courses of DEC do not always succeed in eliminating the signs and symptoms of loiasis in humans. Further work on the chemotherapy of loiasis in monkeys, using DEC and other filaricides, might improve the treatment of this disease.
Effects of multiple monthly doses of ivermectin on adult Onchocerca volvulus.
This paper assesses the effects on adult Onchocerca volvulus of monthly doses of ivermectin (150 micrograms/kg) given over 4, 8, and 12 months to patients in Guatemala. Nodules were removed 4 months after the last dose; the adult O. volvulus were extracted by collagenase digestion, studied by histological techniques, and compared with worms from untreated patients. Twelve monthly doses killed a proportion of the adult worms (12% of males and 22% of females), leaving the remainder relatively unaffected and the females slowly resuming embryogenesis. After 8 and 12 doses, a number of female worms had resumed embryogenesis in 1 genital tract only, and in 1 female a total degeneration of 1 ovary was seen. Ivermectin also led to a marked drop in the number of male worms in nodules. No serious adverse reactions occurred and the treatment was well accepted.
An improved method of examining adult Onchocerca volvulus worms.
Adult Onchocerca volvulus worms were extracted from alcohol-fixed nodules by collagenese digestion. The worms were stained in Mayer's haematoxylin and brought up into glycerol. In this viscous fluid the stained and cleared worms could be unravelled in sequential lengths of 4-6 cm and mounted in glycerol on slides for longitudinal examination of their structure. The worm lengths were then cut into small pieces, 0.5 cm long, which were implanted sequentially into slits in a brick-shaped block of formalin-fixed calf's brain (white matter) also in glycerol. The brain blocks were then processed in paraffin wax and cut as 6 microns histological sections, enabling transverse sections of the worm, stained as required, to be examined in sequential order.
On the reproductive activity of the female Onchocerca volvulus.
Onchocerca volvulus worms, extracted from nodules by collagenase digestion, stained with haematoxylin and cleared in glycerol, were unravelled for longitudinal examination and later embedded in brain blocks for study of serial transverse sections. A classification system for female worms is proposed, based on the reproductive status of 446 worms from Guatemala, 94 from Liberia and 125 from Mali. They were categorized into fecund, inseminated specimens; uninseminated, but potentially fertile specimens, shedding ova destined to degenerate; worms changing from the uninseminated to the inseminated state and vice versa, which were few in number; old worms, with degenerate ovaries, whose genital tracts were either empty or had disappeared; and moribund or dead worms, characterized by loss of turgor, collapse and degeneration, calcification, or invasion by polymorphic, basophilic cells. Potentially fertile worms shed oocytes continuously and, when they were inseminated, embryonic development ensured. No evidence was found of a periodic cycle of reproduction. Inseminated worms were found in nodules without a male worm, and uninseminated worms in nodules harbouring male worms. Measurements are recorded of portions of the female reproductive tract and of the length of uterus occupied by the various embryonic stages in fully fecund worms. A significant difference in the length of the body behind the first and second ovaries was observed as between worms from West African savanna (Mali) and forest (Liberia). Limited observations were also made on meiosis in the oocyte, penetration of the oocyte by sperm, formation of the ovum, syngamy and zygote formation.
Human onchocerciasis--an overview of the disease.
The general characteristics of Onchocerca volvulus infection and its transmission are outlined in this overview of human onchocerciasis. The pathogenic role of the microfilariae, producing lesions of the skin, lymphatic system, eye and deep organs, are described, along with the main clinical manifestations of the disease. The global prevalence and distribution of onchocerciasis are given. Best estimates in 1985 gave 86 million persons at risk, 17.8 million infected, 336,400 blind and a like number suffering from severe visual impairment. The vast majority was in Africa. The impact of onchocerciasis on communities in the Sudano-Guinean savanna zone of Africa is outlined, emphasizing the very high blindness rates and the increased mortality among the blind. Communities so affected cannot remain economically viable. They are forced to desert their villages and the fertile land near rivers. The background to the establishment of the Onchocerciasis Control Programme in West Africa (OCP) is given and the successful 10-year results of this campaign, which is based on prolonged, regular Simulium larviciding, are outlined. In the context of the future of the OCP and of the control of onchocerciasis elsewhere in the world, the need for improved chemotherapy is discussed. The prospects for large-scale suppressive therapy have greatly improved following the registration of ivermectin in 1988 for use in human onchocerciasis. The potential and possible uses of this drug, as a single-dose, non-toxic microfilaricide, which excites very little Mazzotti reaction and has a prolonged microfilarial suppressant action, are discussed. It is considered that an effective non-toxic macrofilaricide is still a prime need for onchocerciasis control.
Onchocerciasis: a review of clinical, pathologic and chemotherapeutic aspects, and vector control program.
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Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen.
Adult Onchocerca volvulus were isolated from nodules removed from onchocerciasis patients at four locations--two in the West African Sudan-savanna region (near Bamako, Mali, and Touboro, Cameroon), one in a West African forest region (Kumba, Cameroon) and one near Guatemala City, Guatemala. Four different cDNA expression libraries were constructed in bacteriophage lambda gt11 using poly(A)+ RNA from the adult female worms. Individual cDNA clones of single copy genes were used to compare the genomes of parasites from the different locales and to show that the haploid genome of O. volvulus is 1.5 x 10(8) base pairs. About 1 in 700 recombinant clones in each of the four amplified cDNA libraries produces a fusion protein recognized by pooled human anti-O. volvulus antisera. Partial sequence determination of a 2.0 kb cDNA clone for an O. volvulus protein that induces an immunodominant response in rabbits revealed that this antigen has sequence similarities with Caenorhabditis elegans myosin and with schistosome paramyosin (which confers partial protection against schistosome infection). The four cDNA libraries have been deposited with American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., for general distribution under ATCC Number 37509.
Special requirements for use of adult Onchocerca volvulus in molecular biology.
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Attempts to infect jirds (Meriones unguiculatus) with Wuchereria bancrofti, Onchocerca volvulus, Loa loa loa and Mansonella ozzardi.
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