Wolbachia endobacteria depletion by doxycycline as antifilarial therapy has macrofilaricidal activity in onchocerciasis: a randomized placebo-controlled study

Abstract

In a randomized, placebo-controlled trial in Ghana, 67 onchocerciasis patients received 200-mg/day doxycycline for 4-6 weeks, followed by ivermectin (IVM) after 6 months. After 6-27 months, efficacy was evaluated by onchocercoma histology, PCR and microfilariae determination. Administration of doxycycline resulted in endobacteria depletion and female worm sterilization. The 6-week treatment was macrofilaricidal, with >60% of the female worms found dead, despite the presence of new, Wolbachia-containing worms acquired after the administration of doxycycline. Doxycycline may be developed as second-line drug for onchocerciasis, to be administered in areas without transmission, in foci with IVM resistance and in areas with Loa co-infections.

Fig. 1

Flow chart of patients who took part in the study. ^a Patients who had received ivermectin (IVM) at the 6 months point of time. ^b Only patients who had received ivermectin at 6 months were analysed. The total numbers comprise the patients who underwent nodulectomy and had received ivermectin (see footnote ^a) plus patients who were only available for skin snipping (but not for nodulectomy) at the respective point of time. ^c Four additional patients (two from each of the doxycycline groups), three of whom were not available for nodulectomy between 6 and 27 months, were nodulectomised at 39 months, increasing the total number of nodulectomised patients to 14/22 and 19/22 in the 4 and 6-week groups, respectively

Fig. 2

Differentiation between living and dead O. volvulus worms by immunohistology. “Living” is defined as alive before fixation of nodules. a At left, a living male worm labelled red (arrowhead) and at right, a dead female with strongly degenerated organs after doxycycline and ivermectin. b Detail of the same slide as (a) but from another region showing the red labelling of the epithelium of the testis by antiserum against APR (open arrowhead) and of the median chord (closed arrowhead) of the living male in contrast to the dead female (arrow). The testis is filled with sperms. c A living male with well-labelled epithelium of the testis (open arrowhead) and hypodermis (closed arrowhead) and an unlabelled dead female (black arrow) from a placebo patient not treated with ivermectin. d A moribund female with a pleomorphic neoplasm in the pseudocoeloma cavity (black arrow) from a placebo patient 14 months after ivermectin. The intense blue staining of the gut indicates the older age of these worms and it hides the red labelling of APR on the photos. All patients were operated 27 months after the onset of the study. Sections were stained with antiserum against a lysosomal aspartic protease (APR) of O. volvulus only labelling the organs of “living” but not of dead filariae. Scale bar = 100 μm

Fig. 3

Living O. volvulus worms acquired before and after doxycycline treatment without and with Wolbachia endobacteria. a At left an old female worm without endosymbionts and at right a young newly acquired female and a male worm with red labelled Wolbachia (open arrowhead). Onchocercoma excised at 27 months. b As in (a), at left an old female and at right new young worms at 20 months (open arrowhead). c Another section of the old female shown in (b)—but from another place of the same slide—without Wolbachia and embryos and with a dark, pigmented gut (closed arrowhead). d Detail of (b) showing both the young female and male worms with endobacteria labelled in red (open arrowheads). The gut is less pigmented (closed arrowhead) and the uterus contains coiled microfilariae (arrow). Sections stained with antiserum against Wolbachia surface protein (DiWsp). Scale bar = 100 μm