This open trial was conducted between 8/2003 and 11/2006 in the Assin district, Central Region of Ghana. This focus lies south of the Onchocerciasis Control Programme in West Africa (OCP) and hence transmission is ongoing. In 1999, a rapid assessment of 30 men older than 19 years had identified many villages in Assin as hyperendemic. In the selected study village Awisem, nodule and mf-carrier rates both were 74% (R. Horstmann and D. W. Büttner, Bernhard Nocht Institute). Awisem is situated 1 km west of the River Pra, where Simulium sanctipauli vectors breed (map and vector epidemiology see Kutin et al. 2004). Ivermectin mass treatment started in Assin in 1999, but due to the remoteness of the village it proceeded at a low coverage until the end of this study (R. Garms, Bernhard Nocht Institute, unpublished report, 2006). A reduction of the infectivity rates and the loads of L3 in Simulium was not observed by Garms and colleagues in 2002 (Kutin et al. 2004) and also not in 2006 (R. Garms, unpublished report, 2006).
Ethical aspects and informed consent
The study was approved by the Ethical Research Committee of the School of Medical Sciences of the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana. The study conformed to the principles of the Helsinki Declaration of 1975 (as revised 1983, 2000, and 2002). To inform the villagers, there was first a meeting held with village elders, where the study procedures and rationale were explained. After general consent obtained by the elders to work in the community, the study procedures were explained to interested people in English by the trial physician and then again in the local Twi language by YMD. Informed consent was obtained from participants and documented by the signatures of two witnesses.
Inclusion and exclusion criteria
Individuals eligible for participation were adult men aged 18–62 years. Women older than 40 years who declared to be in their post-menopause were also included. Participants with a body weight of at least 40 kg, in good health without any clinical condition requiring chronic medication, had to have at least two palpable nodules (onchocercomas) at different sites of the body.
Exclusion criteria encompassed a history of intolerance to doxycycline and of alcohol or drug abuse. Other exclusion criteria were assessed by dipstick chemistry (Reflotron®, Roche, Mannheim, Germany): pregnancy (in case of wrong oral declaration of being post-menopausal), hepatic and renal enzymes above normal values (γ-GT [0–28 IU/L], ALT [0–45 IU/L], and creatinine [53–126 µmol/L]).
Patient treatment and nodulectomies
Twenty-four volunteers were included in the study (Fig. 1). They received 100 mg capsules of doxycycline daily for 5 weeks (Vibramycin®, supplied by Pfizer, Karlsruhe, Germany). Doxycycline was administered after patients had taken a meal. Treatment was monitored daily by a physician (MB). In eight cases of unplanned absence (e.g., due to travelling), the patients were given extra daily doses at the end of the 5 weeks. The maximum of extra doses were three. Two individuals did not complete the treatment. All participants were requested not to take part in ivermectin mass treatments during the study. Fulfillment of this request was checked by questioning. This request was justified since doxycycline leads to long-term sterilization in contrast to ivermectin that causes, after the first doses, only an incomplete and temporary interruption of embryogenesis (Duke et al. 1991, 1999). Thus, the planned doxycycline was expected to supersede the efficacy of ivermectin. Participants received ivermectin from us after the nodulectomies at 27 months (11/2005) and after re-examination at 39 months (11/2006).
At 21 and 27 months after the beginning of doxycycline treatment, nodulectomies were performed aseptically under local anesthesia in the Dunkwa Regional Hospital, as described (Albiez et al. 1988a). Five of the 20 doxycycline patients were operated at both points of time. Wound treatment was begun in the hospital and continued in the village by the trial physician.
In addition to the doxycycline-treated patients, ten patients from the same village, who had not been enrolled in the study and had received neither doxycycline nor ivermectin, were enrolled for nodulectomy at 27 months.
Previously, we had studied the efficacy of 100 mg/day doxycycline administered for 6 weeks (Hoerauf et al. 2000, 2003). The aim of the present study was to investigate the effects of a shortened regimen of only 5 weeks doxycycline treatment on the depletion of Wolbachia endobacteria from O. volvulus, on interruption of embryogenesis and mf production, and to detect macrofilaricidal activity. We deliberately chose to nodulectomize the patients after a longer period of observation than in the preceding study, namely at 21 and 27 months, since we knew from this previous study as well as from unpublished studies (Hoerauf et al. 2003; in preparation) that the depletion of Wolbachia and the interruption of embryogenesis could still be observed up to 27 months but macrofilaricidal activity would become detectable only later than 11 months, e.g., at 18, 21, or 27 months.
The primary outcome measurements were female worm fertility, adult worm survival, and the presence of Wolbachia endobacteria. All parameters were assessed by immunohistology of extirpated nodules. The secondary measurement was the analysis of skin mf from two skin snips that were taken from the doxycycline-treated patients at study onset as well as at 21 and/or 27 months later.
The sample size was planned on the basis of previous histological analyses: accordingly, 50–100 female worms should be available per treatment arm to detect significant differences in embryogenesis and vitality of female worms (Albiez et al. 1988b, Awadzi et al. 1995a, b, 1999; Büttner et al. 1988; Hoerauf et al. 2003). These worm numbers were obtained for both the doxycycline and the untreated group.
Immediately after extirpation in the operation, theater nodules were fixed in 80% ethanol or in buffered 4% formaldehyde solution. They were embedded in paraffin. Smaller nodules were totally embedded. From medium-sized and large nodules, the outer portions were cut from two sides to obtain the middle portion of the nodule on the level of the two largest diameters. These portions were about 2–4 mm thick, depending on the size of the nodule. From a few larger nodules, two portions were embedded. Because of the damage caused by the cutting, the first sections were discarded. Nodules with many calcified worms were examined using a dissecting microscope, deparaffinized, then decalcified using EDTA, and newly embedded. Sections were stained by hematoxylin and eosin, and some sections by Gomori’s method for iron, to differentiate old and young new worms (see below). For immunostaining, the alkaline phosphatase anti-alkaline phosphatase technique was applied according to the manufacturer’s instructions (DakoCytomation, Hamburg, Germany). To demonstrate the presence of Wolbachia, a rabbit anti-serum against Dirofilaria immitis Wolbachia surface protein (DiWsp) was used diluted 1:1,000 (Bazzocchi et al. 2000). Vitality of adult worms, oocytes, and embryos was assessed with rabbit anti-serum against a cathepsin D-like lysosomal aspartic protease of O. volvulus (APR) diluted 1:1,000 (Jolodar et al. 2004). This antibody helped also to differentiate the worms and to count them. As secondary antibody, an anti-rabbit mouse monoclonal antibody was used (clone MR12/53, DakoCytomation). Fast Red TR salt (Sigma, Deisenhofen, Germany) was applied as chromogen and hematoxylin as counter stain (Merck, Darmstadt, Germany).
Assessment criteria were used as described (Büttner et al. 1988; Hoerauf et al. 2003). Ten or more sections from each nodule were independently analyzed by two persons. Counting of different female worms was easy since the average numbers of all females per nodule were only 1.5 for untreated and 1.7 for doxycycline-treated patients. For larger nodules, we used the localization in different worm centers divided by connective tissue, the size of the worms and of their organs, the morphology of the worms, the status of embryogenesis, the pigmentation of the intestine and the uterus muscles, the vitality, and the degree of APR labelling. The characteristics for worm death included completely calcified worm portions, remnants of the cuticle of nearly absorbed filariae, loss of body wall integrity, loss of nuclei, and absence of APR staining (Figs. 2, 4, and 5; Jolodar et al. 2004). Degenerated worms still APR positive were considered moribund (Duke et al. 2002; Gardon et al. 2002) and also classified as “dead”. However, this criterion was only used for five worms with neoplasms (Duke et al. 2002; Fig. 3) and one other worm after doxycycline. ‘Living’ means alive at the time of nodulectomy. All embryonic developmental forms from the stage of two cells to the stretched mf in the uterus were classified as ‘embryos’ (Büttner et al. 1988; Hoerauf et al. 2003). At the time when the nodules were extirpated, 25–45 months had passed since those patients, who had taken ivermectin before the onset of this study, had taken their last dose. It is known that after such a long interval the influence of ivermectin on embryogenesis and mf production is reduced or nearly nil (Albiez et al. 1988b, detailed Duke et al. 1991). For the analyses, the nodules extirpated from doxycycline-treated patients at 21 and 27 months were summarized in Tables 2–5. Regarding the primary objectives addressed in this study, a period of 6 months between the nodulectomies is short and we do not know of any relevant effect of a nodulectomy a few months previously on other adult O. volvulus in the patients nodulectomized twice.
Determination of microfilariae loads
Microfilarial loads of treated patients were assessed before and at 21 and/or 27 months after the start of doxycycline administration. From patients who only attended one of the two follow-up points of time, the available skin mf value was taken for analysis. From those who attended both follow-up times, one value was randomly selected. For mf analysis, two skin biopsies of 1–3 mg were taken from the buttocks using a Holth punch. Each biopsy was immersed in 0.9% NaCl solution in a well of a microtiter plate (Nunc, Roskilde, Denmark). The biopsies were incubated at room temperature for 6 to 20 h. The solution was then transferred onto a slide for microscopic examination. The biopsies were weighed using a Sartorius electronic balance (Göttingen, Germany). Microfilariae per milligram of skin were calculated as median and geometric mean. For the latter, the calculation according to Williams was performed as used by WHO (Remme et al. 1986). Since the geometric mean cannot use zero, the calculation uses mf +1 values, and from the calculated mean, 1 is subtracted afterwards.
The Wilcoxon signed rank test was used to test for a change of mf loads in Table 4. The chi-square test and the Fisher’s exact test were applied for qualitative data. In addition, in order to take into account possible dependencies between the nodules achieved from one patient (as described, e.g., by Duerr et al. 2001), alternating logistic regression, as implemented in the SAS procedure Genmod, was used to analyze the rate of normal embryogenesis, living female worms, and the presence of nodular mf. For the calculations, StatView® software version 4.5 and SAS 9.1 were used.