Report on medical research and proposed programme for 1977

ONCHOCERCIASIS CONTROL PROGRAMI\IE IN THE VOLTA RIVER BASIN AREA ocPfsrAc4.4 SCIEI{TIFIC AND TECHNICAL ADVISORY COMMITTEE ORI GIML : ENGLI SH Fourth meetlng Geneva, 2-4 November 1976 Agenda item 5. (b) REPORT ON MEDICAL RESEARCH AND PROPOSED PROGMMIVTE FOR 1977 Proposals for future research activities on epidemiology, parasitology, irnmunotogy and chemotherapy are presented with a brief review and, where appropriate, with an evaluati.on of research currently carried out under estabtished agreements and of results obtained in the applied research programme which is being undertaken by the Epidemiological Evaluation Unit. A. EPIDEMIOLOGY l. Studies on the d ics of onchocerciasis transmission and on the pa thogen esis of the disease tn a savanna area of northern Cameroon These investigations, which are being conducted by the Medical Research Council, UK, and the University of TUbingen, West Germany, are progressing very satisfactorily. Hyper-, meso- and hypo-endemic villages were selected after preliminary parasitologicat and ophthalmologlcal data had been collected and intensive and continuous entomological observations had been carried out. These villages will be followed up at regular intervals by the medical team, whilst entomological activities will continue without interruption. Further studies undertaken during the year confirned that a high concentration of microfilariae near the eye is a good indication of ocular pathology. Nodules and skin-snips from heavily infected rain-forest patients were shipped in tiquid nitrogen to Dr A. Garner, Institute of Ophthalnology, London, for immunopathological studies on the eyes of the rabbit. Difficulties were experienced with the cryopreservation of onchocerccmata and infected skin-snips, inasmuch as during the journey Cameroon-London microfilarj.ae lost their viability. With more precise instructions regarding cryopreservatlon of the infected material it is likely that the problem can be solved. Progress was made in immunological studies conducted by the British Medical Research Council Team in cooperation with Prof. J. Ngu, "Centre Universitaire pour les Sciences de la Sant6", Yaound6, Cameroon, and Dr Bartlett of the Nuffietd Institute of Cooperative Medicine, London. 2A trial began on 30 patients, who were given a lO-day course of DEC followed irnmediatety by four weekly injections of suramin, to determine whether suramin ls better tolerated when a large number of microfilariae have already been killed by a short course of DEC. Another study proved that Deseril alone is insufficient to alleviate side-reactions due to DEC, and heavity infected patients in north Cameroon could not be treated safely with DEC wrthout corticosteroid cover. The situation drd not improve when DEC and Deseril were administered under Betnelan cover. Drug trials witt soon start wit\ anthelminthic and trypanocidal compounds such as metrifonate (Trichlorphon) arid nifurtimox (Lampit). 2. Histo ical examinatrons on sk lesions due to onchocerciasis Under an agreement with oCP, thd "Istituto di Istologia e Anatomia PatoIogica", University of Rorne, ItaXy, is carrying out hlstological exam- inations on skin fragments taken frorf patients in the Programme area. The study of the histopathologicat changds, compared with photographic material on the same patients, should permit tietter identification and classification of the early skin-lesions due to onchocerciasis. At the same Institute hi-stological examinations for the presence of microfilariae were carried out on 34 specimens of cord tj-ssue from new born babies, and 34 specimens of placentae from their mothers, tn an attempt to determine whether transplacental iinfection occurs in onchocerciasis. 3. Transplacental i.nfectron in onchocerclasis Fotlowing the recommendation wh{ch was made at the third meeting of the Scientific and Technicat Advisory Corlrmittee (2-4 March I976) prelinlnary studies were undertaken by the OCP E$iOemiologlcal Evaluation Unit at Bawku Hospital, in northern Ghana, to inve$tigate whether transplacental transmission is a recurrent phenomenon in onchoce*ciasis. Skin snips were taken from elev8n chltdren born to ten j.nfected mothers, snips were also taken from the placentae and from the umbilical cords of the infants. Positive skin-snips were found in two infants, one of which also had microfilariae in the unbllical cord; microfilariae were also found in the umbillcal cord of a third child. No microfilariae were recovered from the placental snips or from 2 ml. of blood which had been drawn from the placental vessels. From this preliminary experience i-1 would appear that microfilariae of O.volvulus can be transmitted from an i,nfected rnother to the embryo during pregnancy, and that these microfilariae reach the new born without passing from the maternal circulation across the placenta to the foetal circulation, The investigation will be extended in due course: histological examinations for the presence of microfilarlae will be carried out on specimens of placentae, cord tissue, and blood will be taken from mothers, as well as from the cords, for the detection of microfilariae. The positive infants found at Bawku 3hospital will be followed up and the epiderniological significance of this new fi.nding will be assessed. Epidemlologically, however, the demonstration that "in utero" transmission of o.volvulus microfilariae may occur, is extremety important. This implies that whenever positive infants in areas under control operationS are found, they cannot necessarily be related to the persistence of the transmission of the disease. Among 5r,57O examinations already carried out in the Programme area in the course of basic evaluation surveys, positive infants and young children were frequentty encountered, as shown by the following figures: Age O - 5 months 6 - 11 L2-23 Number Registered 1036 664 ro62 Number Examlned 789 6r8 1008 Number Positive 38 29 50 o1 /o Positive 4.8 4.7 5.o 4. Epldemiological stud ies in the Farako area, Sikasso reg ion of Mali where control operations had been carried out without interruption since 1 966 The Epidemiological Evatuation Unit carried out special investigations on the population of Farako area, in the Sikasso region of Mali, to assess the impact of control operations on the prevalence and evolution of the disease. In this area DDT larviciding operations had been carrred out from October 1962 to the end of 1972, with interruptions during the periods of March 1964 to January 1g65 and March 1965 to January 1966. From 1972 onwards, treatment was carried out with Abate. Although the area is retatively sma11, the results were not homogeneous. In originally meso-endemic villages the disease had practically disappeared, whereas in previously hyperendemic villages the prevalence of the disease had been reduced to a 1evel of 3O-4O%. The fly could not be eliminated everywhere and some transmission persisted in a few instances, due either to undetected breeding places which were not treated during the early years of the campaign, or to infected flies coming from adjacent untreated rivers. The rapid regression of the incidence of the disease in villages near the capture stations, where the theoretical biting fIy density had been reduced to a mean value equal or inferior to one thousand flies per man per year, indicated that transmissi.on, when present, was minimal, and that as the reservoir of the infection declines transmission is bound to be further reduced, and possrbly eliminated. Ophthalmological examinations carried out in 1968, 1971 and 1975 in one village of the same focus showed that the ocular infections had consistently diminished, and that the risk of blindness had been greatly reduced. BI 5. Visual screening of illlterate populations Experience from a number of ophthalmological field surveys of illiterate populations in the Prograrnme area in 1975 showed that the E-chart for testing visual acui-ty was not always applicable, because of inability to learn the test and to give reliable responses. A comparison was made between the E-test and the Sjdgren I{and-test as a result of whi.ch the Sjdgren Hand-test, which had gj.ven significantly better results replaced the E-test in routine ophthalmological examinations in the fie1d. 6. Distribution of animal onchocerciasis and identification of infective filarial larvae in S.damnosum Following the recommendation made by the Scienti.fic and Technical Advlsory Committee at its second and third meetings, an Agreenent was established with the "Museum national d'Histoire naturelle", Paris, France, to study the distribution of Onchocerca species in domestic and wi ld animals in the Programme area, and the vectors. These studies, which include investi- gations on the identificatron of infective filarial larvae in S.darnnosum, are due to commence in Ivory Coast in the very near future and will be extended gradually to Togo and Benln. PARASITOLOGY Improvement of the skin-biopsy technique At the third meeting of the Scientific and Technical Advisory Committee it was reported that studies were in progress in the OCP area to compare the respective values of the different parasitological diagnostic techniques at present available and to assess the advantages or disadvantages of each method when applied in the field. The membrane filtration technique' suggested by Dr P. Scheiber, though accurate and precise' appears rather inpractical for routine evaluation work since it is time-consuming. The nethod is, however, being apptied for the study of the distributi-on of D,streptocerca in the Programne area. A comparison was also made between the standard method (i.mmersion of the skin-snip in distilled water and reading after 30 minutes) and the "wel1" technique (immersion of the skin-snips in wells containing saline and reading after 3 hours or more with or wj-thout the addition of a drop of a LO% formalin solution,) The "weII" technique proved to be definitely superio;" to the standard method both in terms of sensitivity (5'6% more positive cases are detected) and in terms of accuracy in measuring microfilarial densities (emergence of about 55-60% more microfilariae than with the standard method)' Substantial vari-ations i-n microfilarial counts are obtained with the standard method if the reading is not carried out within the recommended 30 ninutes, whereas with the "wel1" technique these variations are minimal if the reading is made during a period of 3-24 hours. However' under the prevailing working conditions i,n the field it was found that the "wel1" technique presents more risk of errors than the standard method. some of these errors could be identified and could be related to: (a) faulty handling of the skin-snips inthewellsofthemicrotitrationplatewithresultingconfusionin 4 5identification of subjects; (b) improper washing and cleaning of the schlero- punch after imnersion ln sterllizing liquid. Due to the greeter sensitivity of the "we11" technique which permits detection of more positive cases, and therefore a more accurate prevalence rate of infection, the declsion was taken to adopt the standard method in simple evaluation surveys and to re-exanine all the negative snips using this technique. A code concerning the method being utilized has been added to the sinple survey reporting, form 5602 (i.t already existed in the 560l form for detailed surveys) so that the information is correctly stored in the computer and a clear indication is provided on the method to be utilized in successive surveys. The "wel1" technique is also applied as a routine in all detailed evaluatlon surveys as well as i.n the course of special surveys. Differences in regard to microfilarial densities will continue to persist for the time being depending on the method used, however, an adjustnent for the standard method can be applied and eventually introduced in the village summary tables. Further studies will be carried out on the "we11" technique in order to elininate the error factors occasionally encountered and to render this rnethod as eesy to perforn and as reliable as the standard one. 2 Distribution of Dloetolonema streotocerca in the Programne area The di.stribution of D.strePt ocerca and w.bancrofti in population samples of selected villages in northern Ghana was investigated. So far these two filariat infections have not been detected. Sirnilar exaninations wiIl be carried out systenatically in the different geographical areas of the Programnre. 3 Acid phosphatase activi tv in On s uri crof i lariae The difference in the locatization of acid phosphatase activity in microfilariae of dlfferent species has provided a new taxonomic tool for their differentiation. Strikingty different patterns for acid phosphatase act ivi ty have been shown in Dirofilaria immitis Di peta lonema reconditum, Dirofl laria rcn6ns Brugia pahangi, the sub-periodic form of Brugla malayi as well as in 7 different species of New World monkeys. The Epidexniological Evaluation Unit, in collaboration with the Institut National d'Hygidne, Lom6, Togo, plans to study the histochemical staining pattern of nicrofilariae of O.volvulus and to determine whether microfilariae of the forest-strain can be differentiated from the savanna-strain on the basis of the specific distribution of their acid phosphatase activity. 4. rn vitro culture of nicrofilariae At the Johns Hopkins university, schoot of Hygiene and Public Health, Baltinore, United States of America, microfilariae from an O.volvulus Guatemalan strain have been su(:cessfully cultured in vitro for as long as 60-80 days. Up to 20% and l-5% of the cultured microfilariae developed into "sausage" larvae and "infective-like" Iarvae respectively. The first consign- ment of cryopreserved parasitized tissue from the OCP area to be utilized bfor comparison of the developmental and metabolic characteristics of the West African strain with those of the Guatenalan strain, arrived in non- viable condition. It is planned to continue to provide assistance to this Institute for the study of the metabolism, biochemistry and exoantigens of different strains of O.volvulus microfilariae by in vitro culture methods. As there is sufficient evidence that transport of living specimens from Africa is feasible, further efforts will be made to dispatch more cryopreserved parasitized material from the Programme area to Baltimore. C. IMMUNOLOGY 1. Isolation of the Purified and Specific O.volvulus Antigens The studies on the isolation of speclfic and purified antigens from O. volvulus adult worms, whi-ch have been carried out at the Minerva Institute for Medical Research, Helsinki, Finland, are progressing very satisfactorily. Using modern physico-chemical techniques some of the O,voIvulus antigens were successfully purlfied and one has already been isolated. Purified antigen preparations were also tested in Enzyme Linked Immunosorbent Assay with very promising results. 2 Assessment of a Fluorescent Antibody Test In cooperation with OCP, the Center for Disease Control, Atlanta, United States of America, is assessing the value of an immunofluorescent antibody test using 3 nicron sections of O.volvulus antigens for the serological diag- nosis of onchocerciasis. The preliminary IFA results from sera from Onchocerca patients from the OCP area are now available. The antigen used was a 3 micron formalin fixed, paraffin embedded section of nodules from Guatemala. The results have once again shown that serology, and particularly the IFA test results, do not fully agree with the parasitological conditions of the patients Further tests are envisaged, using as antigen micron sections of nodules from West Africa. The visit to the Programme area of the scientist from the Department of Clinicat Tropical Medicine, London School of Hygiene and Tropical Medicine to test the Enzyme Linked Immunosorbent Assay method in onchocerciasj,s in collaboration wi-th the OCP personnel could not materiali.ze. Attempts will be made by OCP personnel to carry out the test in the Programme area. The possibili,ty of utilizing the puri-fied antj.gen obtained at the Minerva Institute for Medical Research, HeIsinki, Finland will be fully explored. L.I.S.AE3 7D, CHEMOTHERAPY I Studv of the lit erature of suramin and di rbanazind A thorough search and review of the literature on the pharmacodynamics and toxiclty of DEC and suramin was carried out by a consultant, Dr F' Hawking of the Brunel University, school of Biological sciences, uxbridge, uK, with assistance from Professor G, ,Ldmmler of the'Tnstitut f1r Parasitologie", Justus Liebig university, Giessen, Germany, and with the aid of "on-Ii-ne" and "off-line" Medlars searches. The two final documents are being reviewed and will be published in due course. 2. Drug trials at the OCCGE, Centre Mvraz, Bobo-DiouIasso, UPPer VoIta Chemotherapeutic trials are being undertaken at the Parasitological Section of Centre Muraz of the Organisation de Coordination et Coop6ration pour }a lutte contre les Grandes End6mies, at Bobo-Dioulasso in Upper Volta. Two different groups of moderately or heavily infected patients were submitted to treatment with diethylcarbamazine, levamisole, diethylcarbamazine and levamisole adrninistered at the same time. Parasitological, serological, biochenical tests and detailed ophthalmological examinations were carried out before and on completion of the administration of the vari.ous drugs, and will continue at regular intervals. The trials are still in progress but preliminary results seem to indicate the following: (a) diethYlcarbamazine When the drug sas administered for 21 consecutive days, the microfllarial densities greatly reduced and occasionally microfilariae quickly disappeared in skin-snips. The serological and biochemical tests did not show any signifi- cant variation when carried out before and after the adninistration of the drug Side-reactions, e.g. pruritus, joint pains, adenolymphangitis, and in one case a sensible alteration of the general status of the patient, were controlled with antihistamines and corticosteroids. Although a detailed report i.s not yet available, according to infornration received, the microfilarial densities are rapidly buildi.ng uP. (b) levanisole So far there have been no detectable signs that the drug had any effect on the microfitarial densities or on the ocular lesions, when administered at the dosage of zgrfxg for 21 days. (c) diethylcarbamazine and levamisole The addltion of levamisole to diethylcarbamazine seems to have no extra effect either on the microfilarial densities or on the incidence and intensity of the side reactions. The observations will continue and plans were made to start a trial with nifurtimox in the near future. a8 3. Effect on Onchocerca volvulus of various low-total-dose courses of suramin Two agreements were established with the "Unit6 d'Enseignement et de Recherche de M6decine et Sant<i tropicales", Marseille, France, to study the effect of Onchocerca volvulus of vari.ous low-total-dose courses of suramin The first one, which was financed by the Divisj.on of Malaria and other Parasitic Diseases of WHO, involved experiments on rabbits to elicit the comparative blood levels of suramin which develop during these courses. The second agreement, financed out of the UNDP research project for OCP, involves trials of the same dosage schedules on patients with onchocerciasis. These trials are due to commence in Mali outside the Programme area, in the near future. 4. Chenotherapeutic Research Centre at TamaIe, northern Ghana Contractual Technical Agreements will soon be established with the Ghana Government and the Liverpool School of Tropical Medicine. During a recent visit to Ghana, Professor Gilles, Director of the Departmeilt'of Ttopical Medi0ine at the Liverpool School of Tropical Medicine, and,a member of OCP, were able to confirm that a satisfactory chemotherapeutic research centre could be developed at Tarnale Regional Hospital, Discussions took place in Accra with the Director of the Medical Service who expressed the Government's willingness to enter into an Agreement with WHO on the setting-up of the Centre. OCP has recruited a short-term consultant, a laboratory technician who will be in the Programne area in early November, and a technical offj.cer who will be permanently stationed at Tamale as from early November. The first drug trial has been tentatively planned to start 1n early January , lg7'7 . 5. Nodulectomv trial Large-scale chemotherapeutic treatment wi.th the two drugs at present available, namely DEC and suramin, cannot be safely undertaken under the conditions of intensive infection which prevail at present in the Programrne area, due to the severe dnd dangerous reactions associated with the administration of the two drugs. While efforts continue to be made for the improvement of the chemotherapeut treatnent of onchocerciasi,s, OCP will try to find methods which could alleviate the sufferings of the population, especlally with regard to the prevention of the development of eye lesiuns into total Llindness, Negotiations are in progress with the Bernhard-Nocht-Institute, Hamburg, Federal Republic of Germany and one of the ParticipatinBaGEernments, for nodulectomy to be carried out annually over a period of D4 years, in two hyperendemic villages of the Programme area where entomological findings indicate that transmission of the disease has been interrupted following two years of larviciding operations. The aims of this trial are (a) to assess the impact that denodulization may have on the reservoir of the infection where the subjects are no longer exposed to re-infection and (b) to evaluate the effect that this therapy may have on the ocular pathology. I +c I I i dI 6 Aninal model At the third meeting of the scientific and Technical Advisory committee it was reported that an agreement had been established wi th CDC, Atlanta,irds (Meriones C, variipennis whichGeorgia, United States of America, to attempt to infect j unguiculatus) with O.cervicalis. It was intended to use had already been successfully colon O.cervicalis as a source of infecti that of the It iirds which had been of O. cervicalis three had been nec could O.cervicalis be demonstrated. observation. ized and Proven to be a vector of ve material. OCP was recently informed inoculated or exposed to infective larvae ropsied at di,fferent intervals and in none The other eight ii-rds are still under b +