Efficacy of 5-week doxycycline treatment on adult Onchocerca volvulus.

Abstract

The effects of 5-week doxycycline treatment on the depletion of Wolbachia endobacteria from Onchocerca volvulus, on the interruption of embryogenesis and on microfilariae production, and with regard to macrofilaricidal activity were studied. In 2003, in an endemic area in Ghana, 22 onchocerciasis patients received 100 mg/day doxycycline for 5 weeks. Two years after the start of the study, 20 treated and ten untreated patients were nodulectomized and skin microfilariae were counted. The onchocercomas were examined by immunohistology for the presence of Wolbachia, embryogenesis, and vitality of adult filariae. The latter two parameters were further assessed by alternating logistic regression analysis, taking into account the dependency of worms and nodules in patients. Doxycycline resulted in depletion of Wolbachia and in complete interruption of embryogenesis in all worms that were assumed to have been present during treatment. In the treated patients, only 51% of the female worms were alive, compared to 84% in the untreated patients, indicating a moderate but distinct macrofilaricidal activity of doxycycline at this dose. It is concluded that, in areas with ongoing transmission, doxycycline cannot replace regular ivermectin mass treatment because new infections would require repeated rounds of doxycycline. However, doxycycline can be used for the treatment of individuals outside transmission areas, in foci where ivermectin resistance may occur, and in countries where onchocerciasis and loiasis are co-endemic.

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ORIGINAL PAPER
Efficacy of 5-week doxycycline treatment on adult
Onchocerca volvulus
Achim Hoerauf & Sabine Specht &
Yeboah Marfo-Debrekyei & Marcelle Büttner &
Alexander Yaw Debrah & Sabine Mand & Linda Batsa &
Norbert Brattig & Peter Konadu & Claudio Bandi &
Rolf Fimmers & Ohene Adjei & Dietrich W. Büttner
Received: 1 September 2008 /Accepted: 23 September 2008 / Published online: 11 October 2008
# The Author(s) 2008. This article is published with open access at Springerlink.com
Abstract The effects of 5-week doxycycline treatment on
the depletion of Wolbachia endobacteria from Onchocerca
volvulus, on the interruption of embryogenesis and on
microfilariae production, and with regard to macrofilarici-
dal activity were studied. In 2003, in an endemic area in
Ghana, 22 onchocerciasis patients received 100 mg/day
doxycycline for 5 weeks. Two years after the start of the
study, 20 treated and ten untreated patients were nodulec-
tomized and skin microfilariae were counted. The oncho-
cercomas were examined by immunohistology for the
presence of Wolbachia, embryogenesis, and vitality of adult
filariae. The latter two parameters were further assessed by
alternating logistic regression analysis, taking into account
the dependency of worms and nodules in patients. Doxycy-
cline resulted in depletion of Wolbachia and in complete
interruption of embryogenesis in all worms that were
assumed to have been present during treatment. In the
treated patients, only 51% of the female worms were alive,
compared to 84% in the untreated patients, indicating a
moderate but distinct macrofilaricidal activity of doxycycline
at this dose. It is concluded that, in areas with ongoing
transmission, doxycycline cannot replace regular ivermectin
mass treatment because new infections would require
repeated rounds of doxycycline. However, doxycycline can
be used for the treatment of individuals outside transmission
areas, in foci where ivermectin resistance may occur, and in
countries where onchocerciasis and loiasis are co-endemic.
Abbreviations
APR
DiWsp
L3
mf
O. volvulus aspartic protease
D. immitis Wolbachia surface protein
infective larvae of O. volvulus
microfilaria (e)
Introduction
Onchocerciasis is endemic in many sub-Saharan countries
and in minor foci in Latin America and Yemen (WHO
1995b; Boatin and Richards 2006). The number of
Parasitol Res (2009) 104:437–447
DOI 10.1007/s00436-008-1217-8
A. Hoerauf (*):S. Specht:S. Mand
Institute for Medical Microbiology,
Immunology and Parasitology, University of Bonn,
Sigmund-Freud-Str. 25,
53105 Bonn, Germany
e-mail: hoerauf@parasit.meb.uni-bonn.de
Y. Marfo-Debrekyei:A. Y. Debrah:L. Batsa
Kumasi Centre for Collaborative Research in Tropical Medicine
(KCCR),
Kumasi, Ghana
M. Büttner:N. Brattig:D. W. Büttner
Bernhard Nocht Institute for Tropical Medicine,
Bernhard-Nocht-Str. 74,
20359 Hamburg, Germany
P. Konadu:O. Adjei
School of Medical Sciences,
Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana
C. Bandi
DIPAV—Sezione di Patologia Generale e Parassitologia,
University of Milan,
20133 Milan, Italy
R. Fimmers
Institute for Medical Biometry, Informatics, and Epidemiology,
University Clinic, Bonn,
53105 Bonn, Germany

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infections has recently been re-estimated to be more than
37 million (Basañez et al. 2006). The adult filariae, which
have developed from the third stage larvae (L3) transmitted
by blackflies (Simulium), have an average life expectancy
of 10 years (Habbema et al. 1990) and produce millions of
microfilariae (mf). The mf cause dermatitis, skin atrophy,
and inflammation in the eyes, leading to reduced vision and
blindness. Apart from blindness, a high mf load has been
identified as a factor reducing the life span of infected
people (Little et al. 2004). Vector control and mass
treatment with ivermectin have been successfully used to
regionally reduce the burden of this neglected disease.
Current elimination programmes in America and Africa
are based on ivermectin mass treatment. Ivermectin has
strong microfilaricidal and temporarily sterilizing activity
(Awadzi et al. 1999). It has to be administered for many
years until all adult filariae are sterile or dead, because the
worms resume their fertility after interruption of ivermectin
treatment. After administration of frequent doses of ivermec-
tin every 3 months, moderate macrofilaricidal activity is
observed (Duke 2005; Gardon et al. 2002; Kläger et al.
1993). However, the Conference on the Eradicability of
Onchocerciasis (Dadzie et al. 2003) concluded that eradica-
tion would not be achieved at coverage rates of 65% for 35
or more years. Further, it has been reported that some
populations of the intestinal nematode Haemonchus con-
tortus in sheep have developed a certain degree of ivermectin
resistance (Coles et al. 2005). Since genetic selection of low
fertile Onchocerca volvulus by ivermectin treatment has been
observed, it was hypothesized that O. volvulus populations
more tolerant to ivermectin may also be selected (Bourguinat
et al. 2007). Epidemiological hints for a suboptimal perfor-
mance of ivermectin against O. volvulus populations have
been detected in northern Ghana (Awadzi et al. 2004a,b;
Osei-Atweneboana et al. 2007). Therefore, alternative drug
regimens must be identified before ivermectin resistance may
develop and spread. It has been a long-standing aim to
develop a safe macrofilaricidal or permanently sterilizing
drug.
Doxycycline has been introduced as a novel chemother-
apeutic principle, targeting Wolbachia endobacteria in the
worms (Hoerauf et al. 2000, 2001, 2003). Given for 6 weeks
at 100 mg/day, doxycycline led to Wolbachia depletion
and sterilization of female filariae up to 18 months. There
was also a reduction in the proportion of living female and
male worms at this point of time, but the number of
extirpated worms was too small to allow the definite
conclusion of a macrofilaricidal effect. However, a macro-
filaricidal activity of doxycycline was demonstrated
against Wuchereria bancrofti (Taylor et al. 2005; Debrah
et al. 2006b), and recently also against O. volvulus, albeit
at a higher daily dose of 200 mg/day, also after a longer
observation period (Hoerauf et al. 2008).
Therefore, the present study was conducted focusing on
the assessment of a macrofilaricidal and long-term steriliz-
ing activities after the longer observation time of 21 and
27 months compared to the earlier study (Hoerauf et al.
2000, 2001, 2003). Second, since we knew that a
doxycycline treatment shorter than 6 weeks also depletes
Wolbachia (Debrah et al. 2006a), the regimen was reduced
to 5 weeks at only 100 mg/day of doxycycline. Another
aim was to analyze the efficacy of doxycycline alone
without administration of ivermectin during the trial. Here
we report on an open trial in Ghana, comparing treatment
with doxycycline at 100 mg/day for 5 weeks against a
control group without treatment.
Materials and methods
Study area
Thisopentrial was conducted between8/2003and 11/2006in
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 epide-
miology 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 Commit-
tee 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.
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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 intoler-
ance 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 moni-
tored 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 individ-
uals 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 doxycy-
cline nor ivermectin, were enrolled for nodulectomy at
27 months.
Objectives
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.
Outcome measurements
The primary outcome measurements were female worm
fertility, adult worm survival, and the presence of Wolba-
chia endobacteria. All parameters were assessed by
immunohistology of extirpated nodules. The secondary
measurement was the analysis of skin mf from two skin
Patients with > 1 nodule
selected for doxycycline
during survey in 8/2003 (n = 28)
Included in study (n = 24)
Received doxycycline
(100 mg / day) for 5 weeks
Completed treatment (n = 22)
Dropped out (n = 2)
Nodulectomy in 5/2005 (n = 12)
Of these, completely
nodulectomised (n = 7)
Not selected (n = 10)
Nodulectomy in 11/2005 (n = 13)
Of these, re-operated (n = 5)
Refused nodulectomy (n = 2)
Examined by histology (n = 20)
Excluded for not meeting
inclusion criteria (n = 4)
Patients with > 1 nodule
selected as controls during
survey in 11/2005 (n = 10)
Nodulectomy in 11/2005 (n = 10)
Refused nodulectomy (n = 0)
Examined by histology (n =10)
Fig. 1 Trial profile of patients
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snips that were taken from the doxycycline-treated patients
at study onset as well as at 21 and/or 27 months later.
Sample size
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.
Immunohistology
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 objec-
tives 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 (Göttin-
gen, 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
440Parasitol Res (2009) 104:437–447

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cannot use zero, the calculation uses mf +1 values, and
from the calculated mean, 1 is subtracted afterwards.
Statistical methods
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.
Results
Participant flow
Two of the enrolled 24 patients were excluded because they
were absent too often during treatment (Fig. 1). Two
patients who had completed the doxycycline treatment
Fig. 2 Signs of certain death
are entirely calcified worm por-
tions (arrowhead) or small cu-
ticular fragments of nearly
absorbed filariae (arrows)
Fig. 3 Moribund worms with
pleomorphic neoplasms as de-
scribed by Duke et al. (2002)
(arrowhead) will definitely die
even when the APR-positive
posterior ends appear undam-
aged (arrows). Untreated patient
Fig. 4 A certainly dead APR-
negative worm without organs
and surrounded by a strong
cellular reaction (arrowhead)
and a living filaria with an APR-
positive intestine (arrows) and
degenerated embryos in both
uterus branches (u)
Fig. 5 A dead APR-negative
worm with disintegrated organs
(arrowhead) and a living APR-
positive filaria (arrow) with
empty uterus branches (u). All
images except Fig. 3 from
patients treated 5 weeks with
100 mg/day doxycycline.
Immunohistology using serum
against a lysosomal aspartic
protease (APR). Scale bar=
100 µm
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refused nodulectomy. All palpable nodules of the remaining
20 doxycycline and ten untreated patients were excised and
analyzed by histology. One year after the last nodulec-
tomies, 12 of the 20 doxycycline patients had no palpable
nodules, five had tiny new nodules, and only one patient
had one old nodule on the knee that had not been
extirpated. Therefore, we consider the examined nodules
as representative for these patients.
Baseline data
The characteristics of those nodulectomized patients, who
had completed the doxycycline trial or were included as
untreated patients, are shown in Table 1. Among the 22
doxycycline-treated participants, 14 declared that they had
taken one dose of ivermectin previously; three patients had
taken two doses. These patients had taken the last dose(s)
of ivermectin four (n=2), eight (n=1), 18 (n=11), or 6 and
18 months (n=3) before enrolment. Five doxycycline-
treated and all untreated patients had never taken ivermec-
tin. This prior ivermectin intake had affected skin mf loads
(Table 1, see different mean mf load in the groups with and
without prior ivermectin) However, since the patients had
taken ivermectin only once or twice, this drug intake is not
considered to have disturbed female worm fertility after
another 21–27 months (Gardon et al. 2002; Duke 2005), the
times when the nodules were ectomized and analyzed in
this study. Therefore, it is concluded that a combined
analysis of all doxycycline patients is justified.
Skin manifestations of onchocerciasis were mild. Two
individuals had leopard skin.
Effect of doxycycline on Wolbachia
For Wolbachia assessment, worms were classified as having
many, few, or no bacteria (Table 2). In the untreated patient
group, only in 2% of living females and in 14% of male
worms no endobacteria were detected. In contrast, in
nodules from doxycycline-treated patients, 76% female
and 62% male worms did not contain Wolbachia. However,
we did observe 16% of living females and 16% of living
males with many bacteria. While we do not formally prove
it here, we assume that most or all of the worms with many
Wolbachia had been acquired after the end of the
doxycycline treatment, since they were juvenile or showed
all characteristics of young worms. Also, an overall 10%
replenishment of worms per year can theoretically be
assumed (Schulz-Key and Soboslay 2000; see details in
the Discussion section).
Effect of doxycycline on filarial embryogenesis
and microfilarial load
Table 3 shows the counted, raw data for the worms and
nodules (i.e., not the calculated means±confidence
interval obtained through multiple logistic regression, as
in Table 7). Accordingly, in the untreated group, 45%
living female worms showed normal embryogenesis and
34% of the nodules contained living mf in the host
tissue. In contrast, after doxycycline, only 7% of the
worms presented with normal embryogenesis and 7% of
the nodules had living mf in the human tissue. These
mf were found only adjacent to young productive
Wolbachia-positive worms, which had probably been
acquired after the end of doxycycline during the follow-
up period.
Alternating logistic regression analysis was performed
to take into account a possible dependency between
nodules in patients. The frequencies estimated with this
procedure (Table 7) are only slightly different from
Table 3. Significant differences are found between
untreated patients and patients treated with doxycycline.
Table 1 Characteristics of patients who completed 5 weeks of doxycycline treatment
Characteristics
Baseline dataa
Not treated
No ivermectin
Doxycycline
No ivermectin
Doxycycline
Ivermectin before treatment
No. of individuals
Sex ratio, m/f
Mean age, years
Mean weight in kg
Median of nodules
No. of mf carriers
Median of mf/mg
Ivermectin before recruitment months, meanb
No. with complete treatment
Less days of treatment
10
5/5
34 (18–53)
54 (40–60)
3 (2–7)
10
55 (5–597)
None
Not applicable
5
5/0
35 (25–49)
56 (46–65)
4 (3–5)
4
51 (0–127)
None
5
17
11/6
41 (25–60)
56 (44–68)
4 (2–10)
14
3 (0–121)
14 (4–18)
16
1× only 34 days
aNumbers in brackets denote the range
bThree persons received ivermectin 6 and 18 months before recruitment
442 Parasitol Res (2009) 104:437–447

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The decrease of the skin mf loads is shown in Table 4.
Skin mf levels were determined before doxycycline
treatment and at 21 and 27 months thereafter. 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. Thus, data from 21 doxycycline-
treated patients were collected. According to this analy-
sis, 13 of the 21 doxycycline-treated patients had no mf
after 21/27 months and the mf load had decreased to
0.2 mf/mg skin, supporting an interruption of embryo-
genesis by doxycycline alone since the last ivermectin
intake was by then 31–43 months ago. Since transmis-
sion in this area was not interrupted (R. Garms, Bernhard
Nocht Institute, unpublished report, 2006), we assume
that the producing worms may have been acquired after the
end of doxycycline treatment (see ‘Study area’, Materials
and methods section, and Discussion section). The data
therefore suggest that doxycycline treatment has led to a
complete interruption of embryogenesis in those worms
that had been present at the time of drug administration.
Macrofilaricidal activity of doxycycline
Dead and living worms were assessed using the criteria
described in the Materials and methods section (Figs. 2, 3,
4, 5) and discussed in the Discussion section. Table 5
shows the detailed data for the dead worms. Nodules from
untreated patients contained 16% dead female and 5% dead
male worms. Four moribund female worms with pleomor-
phic neoplasms, as described by Duke et al. (2002) and
Gardon et al. (2002), were included in the category ‘dead’.
The proportions of dead worms are in accordance to those
from other studies in West Africa (Table 6). These data, in
comparison with the data from other endemic areas
throughout Africa, suggest a high and constant degree of
regulation in acquisition and decay of worms, which is
taking place in a very similar way in different O. volvulus
strains from different endemic areas. In contrast, nodules
from doxycycline-treated patients contained a significantly
higher proportion of dead worms, as 49% dead females and
27% dead males were observed. Of the dead females after
doxycycline, 72% were calcified and/or nearly absorbed,
27% were diagnosed as dead based on morphological
criteria or APR-negative reactions, and only one was
moribund.
Results from alternating logistic regression analysis are
shown in Table 7. In the untreated group, 85% of nodules
contained living female worms, as opposed to 56% of
nodules from doxycycline-treated patients. When the
doxycycline subgroup without ivermectin before the start
of the study (see Table 1) was analyzed separately, only
Table 2 Effects of 5 weeks doxycycline treatment on Wolbachia endobacteria
Treatment group No. of patients/nodulesNo. of living female worms No. of living male worms
All With Wolbachia AllWith Wolbachia
Many Few NoneMany FewNone
Not treated
Total for doxycycline
10/41
20/96
51
83
46
13
4
7
1
63a
20
37
14
6
3
8
2
23a
aSignificant difference between the total doxycycline and the untreated group regarding the distribution between many, few, and no Wolbachia
(p<0.0001, chi-square test)
Table 3 Effects of 5-week doxycycline treatment on embryogenesis and production of microfilariae
Treatment group No. of patients/nodules No. of living female wormsNo. of nodules
All Embryogenesis All 00
NoneInter-rupted Normala
Not treated
Total for doxycycline
10/41
20/96
51
83
19
46
9
31
23
6b
41
96
14 (34%)
7 (7%)c
a“Normal embryogenesis” refers to the younger embryos. Degenerated stretched mf assumed to be due to ivermectin prior to study onset are not
considered
bSignificant difference between the doxycycline and the untreated group regarding the frequency of interrupted and normal embryogenesis (p<
0.0001, chi-square test)
cSignificant difference between the doxycycline and the untreated group regarding the proportions of nodules with intact mf (p<0.0001, chi-
square test)
Parasitol Res (2009) 104:437–447 443

Page 8

40% in the group without previous ivermectin were
calculated to harbor living females (not shown). A
significant difference to the untreated group was seen
compared to the doxycycline subgroup without prior
ivermectin, indicating that the macrofilaricidal effect ob-
served was due to doxycycline and not to a potential effect
of an ivermectin intake several years before nodulectomy.
Adverse reactions of doxycycline
Doxycycline treatment was well tolerated by the partic-
ipants. None of them complained or presented severe
adverse reactions such as bloody diarrhea. The dipstick
tests for liver enzymes and creatinine, repeated after 3 weeks
of treatment, remained normal. For a generalized judgment
on doxycycline safety, the study group was too small.
Discussion
In the present study, three new findings were observed: (1)
Wolbachia depletion and interruption of embryogenesis was
achieved after reduction of the treatment duration from 6
(Hoerauf et al. 2003) to 5 weeks at 100 mg/day of
doxycycline. (ii) Wolbachia depletion and sterility were
observed for a longer time of 21 and 27 months than before
and also without accompanying ivermectin treatment. (iii)
Doxycycline showed a macrofilaricidal activity.
The results in Tables 2, 3, and 5 are presented after
analysis per worm or per nodule (Table 3, right column).
This analysis was done to make the results comparable to
earlier studies from this (Albiez et al. 1988b; Büttner et al.
1988; Hoerauf et al. 2003, 2008) and other groups (e.g.,
Awadzi et al. 1995a, b, 1999). It also facilitates tracking
down real individual worms in their different read-out
characteristics (Wolbachia content, embryogenesis, mf in
the human nodule tissue, and vitality), something that is not
possible with logistic regression, because numbers are not
counted but calculated. However, in order to account for the
issue of interdependency of worms in nodules (e.g.,
aggregation as described by Duerr et al. 2001) and nodules
in patients, we used alternating logistic regression to
compare the rate of nodules with normal embryogenesis,
the rate of nodules with mf, and the rate of nodules with
living and dead female worms (Table 7). It confirmed the
analysis from Tables 2, 3, and 5. Interestingly, the macro-
filaricidal effect in the patients without prior ivermectin (see
Table 1) appeared to be even higher (only 40% nodules with
living female worms, data not shown) than in the group that
had received prior ivermectin (61%). Together with the
finding that only multiple, frequent doses of ivermectin
(which were not administered here) show only a moderate
macrofilaricidal effect (Duke 2005), our findings allows the
conclusion that doxycycline is macrofilaricidal on its own.
In addition to interdependency, the dynamics of new
infections of the study patients is another variable that has
Table 4 Microfilarial loads in skin snips before and 21 or 27 months after doxycycline treatment
Treatment groupTime of skin
snip
Months after study
onset
No. of patients
examined
No. of mf
carriers
mf levels (mf/mg)
GMIa
(Range)Medianp value
Not treated
Doxycycline treated
11/2005
8/2003
5 or 11/2005
27
0
21 or 27
10
22
21
10
18
8
43
6
0.9
(5–597)
(0–127)
(0–58)
55
4
0.2
n.a.
0.024b
0.005c
aGeometric mean intensity (GMI) was calculated using the log (x+1) method according to Williams [Remme et al. 1986]
bChanges in median skin mf levels from baseline (at 0 months) to 21 or 27 months were assessed by Wilcoxon signed rank test (in cases where
patients attended both time points, one value was randomly selected)
cDifference in the proportion of skin mf carriers before doxycycline treatment and at 21 or 27 months (Fisher’s exact test)
Table 5 Macrofilaricidal activity of 5-week doxycycline treatment
Treatment group No of
patients
No. of all female wormsNo. of all male worms
AllDead no.
(%)
Calcified or almost
absorbed
AllDead no.
(%)
Calcified or almost
absorbed
Not treated
Total for doxycycline
10/41
20/96
61
162
10 (16%)
79 (49%)a
6
57
21
48
1 (5%)
13 (27%)a
1
5
aSignificant difference between the doxycycline and the untreated group regarding the proportion of dead female worms (p<0.0001, chi-square
test) and dead male worms (p<0.034)
444Parasitol Res (2009) 104:437–447

Page 9

influenced the reported numbers of living and fertile female
worms. Obviously, ongoing transmission leads to the
acquisition of new worms after the administration of
doxycycline, some of which in our study have already
become fertile during the 21–27-month observation period.
As detailed in the Materials and methods section, no
reduction of transmission compared to an area without
onchocerciasis control was observed in two surveys in 2002
(Kutin et al. 2004) and again in 2006 (R. Garms, Bernhard
Nocht Institute, unpublished report, 2006). In summary, in
the latter survey, infection pressure was still at an average
of at least one infectious bite per person per week, or at
least 50 L3 per year. Other factors to consider in the
dynamics of new infection are that the calculated average
adult worm life span is 10 years (Habbema et al. 1990) and
that in an area with ongoing transmission the adult worm
loads are rather stable in an adult patient (Duerr et al.
2003). Worm loads are maintained despite a very large
observed variance of L3 infection pressures, which range
over 100-fold when different areas are compared, so that
between 0.1% and 10% of L3 were calculated to develop
into adult worms (Schulz-Key and Soboslay 2000).
Therefore, it is reasonable to assume a yearly turnover of
approximately 10% of the total adult worm load in areas
with normally ongoing transmission like in our study area.
In the present study, 16% of female worms displayed high
wolbachial loads after 21 and 27 months (“many Wolba-
chia”, Table 2). This number is within the expected range
of newly acquired worms after a 2-year observation period,
and the infection rate with L3 estimated for this area can
maintain such a level of re-infection (i.e., one to two adult
worms per person per year, from at least 50 transmitted L3).
In addition, the female worms harboring many Wolbachia
showed the characteristics of young worms that had been
newly acquired, such as having a small diameter, thin cuticle,
prominent lateral cords and somatic muscles, lack of
inclusion bodies or other signs of degeneration in hypoder-
mis, muscles, and epithelia, many of them being still
nulliparous (Büttner et al. 1988; Franz 1988; Schulz-Key
1988; Duke 1991; Hoerauf et al. 2003). They also showed
little or no brown or (using Gomori’s method) blue staining
in the gut because of less iron deposition (Wildenburg and
Henkle-Dührsen 1999). Therefore, we assume that these
worms have been acquired after the end of doxycycline
treatment. However, this is not critical for the overall
message of this study, as discussed in the next paragraph.
In addition to those worms being nulliparous, the other
worms from this probably newly acquired group showed
normal embryogenesis and fully encompassed the 7% (6/83)
female worms in the doxycycline groups observed with
normal embryogenesis in Table 3.
One can further conclude that, if those probably newly
acquired worms had been subtracted from the data for
Tables 5 and 7, the macrofilaricidal effect of the 5-week
regimen of doxycycline would have been higher than the
counted 49% (Table 5), and vice versa that fewer than the
Table 6 Proportion of dead O. volvulus after 5 weeks of doxycycline and in untreated patients as observed in several studies in West Africa
Treatment/countryNo. of patients/nodules No. of all female wormsNo. of all male wormsReference
AllDead in %AllDead in %
Doxycycline
Untreated
Untreated West Ghana
Untreated East Ghanaa
Untreated, Burkina Gaoua
Untreated, Burkina Diebougou
Untreated, Liberia
20/96
10/41
29/85
25/107
33/217
22/162
16/62
162
61
202
255
500
242
115
49%
16%
15%
26%a
6%
20%
20%
48
21
91
79
207
143
69
27%
5%
3%
4%
4%
4%
0%
This study
This study
Hoerauf et al. 2003
Awadzi et al. 1995b
Büttner et al. 1988
Büttner et al. 1988
Büttner et al. 1988
aIncreased proportion of dead female worms after 2 years of vector control in 1988
Table 7 Multiple logistic regression analysis
Treatment groupFemales with normal embryogenesis Nodules with mfNodules with living females
% (CL)p value % (CL)p value % (CL)p value
Not treated43.5 (27.1–61.4)36.1 (19.1–57.5) 0.005a
0.078b
0.003a
84.8 (59.1–95.6)
Total for doxycycline7.9 (3.7–16.4)<0.0001a
8.3 (3.6–17.0)56.3 (43.1–68.8)0.046a
aNot treated against doxycycline treated
Parasitol Res (2009) 104:437–447445

Page 10

calculated 56% of nodules (Table 7) would have contained
living female worms. However, it is also clear that, after
subtraction of these newly acquired worms, the macro-
filaricidal activity would not exceed approximately 60% in
female worms and thus, doxycycline has not killed all
doxycycline-treated worms at this dose. But also a macro-
filaricidal effect of less than 100% is meaningful for the
patient. For example, suramin does not kill all worms; 34%
of 101 female worms or 0.87 females per nodule were still
alive 12 months after a full course of suramin in an area
without ongoing transmission (Awadzi et al. 1995b)
compared to 51% of 162 living female worms or 0.86
females per nodule after 5 weeks of doxycycline. Further-
more, WHO accepts cure rates of 60–90% for schistosomes
(WHO 1995a). Still, more studies are needed to define the
optimal macrofilaricidal regimen of doxycycline.
The effects of doxycycline on the mf densities were
found to be slow compared to ivermectin and diethylcar-
bamazine. This feature is important when potential adverse
reactions due to the antifilarial activity of doxycycline are
considered. The decay of mf induced by doxycycline is
apparently too slow to induce severe adverse reactions.
Similarly, the macrofilaricidal activity of doxycycline is
slow and does not show characteristics of fast killing of
adult worms, such as nodule perforation as observed after
suramin (Büttner, unpublished). The general adverse reac-
tions to doxycycline, such as bloody diarrhea, are known
from numerous applications, because the drug has been
registered and widely used since long time. Serious adverse
effects were found to be very rare, so that doxycycline is
being used already for other indications by populations in
onchocerciasis-endemic areas, even in those with a low
standard of medical care. Doxycycline is readily available
in pharmacy shops in these areas.
The long doxycycline treatment, the slow decay of mf,
and the appearance of newly acquired worms after
doxycycline in a focus with ongoing transmission show
clearly that ivermectin remains the drug of choice for the
areas which are currently covered by the African
Programme for Onchocerciasis Control (Boatin and
Richards 2006), because doxycycline would have to be
frequently re-administered to people that have already been
treated in order to cure new infection. Indications for
doxycycline have been described recently in detail (Hoerauf
and Pfarr 2007; Hoerauf 2008). They apply to onchocerci-
asis patients who are no longer exposed to re-infection,
because they left the endemic area. Further, in the USA, the
elimination of onchocerciasis has reached ‘end-game’
scenarios, where transmission has apparently been inter-
rupted and only a limited number of people are infected
with onchocerciasis in few foci. Administration of doxycy-
cline to these people has been suggested (WHO 2007) on
the basis of its long-term interruption of embryogenesis.
The new data on the macrofilaricidal activity may further
underscore this argument.
Doxycyclinemayalsobedevelopedasa reservedrugifthe
concerns regarding the potential development of ivermectin
resistance (Bourguinat et al. 2007; Osei-Atweneboana et al.
2007) are proven to be correct and quick action is needed to
prevent spreading of resistant worms. Finally, anti-wolba-
chial chemotherapy may become an option for areas that are
co-endemic for loiasis and where ivermectin treatment has
resulted in the occurrence of encephalopathy (Thomson et al.
2004). Studies in co-endemic foci are currently being
performed.
Acknowledgements
Health Management team of Assin district for their cooperation, and
Dr. John Larbi and Daniel Tagoe, Kumasi, and Ingeborg Albrecht,
Hamburg, for their technical assistance. Pfizer Inc., Karlsruhe,
Germany, generously provided Vibramycin®. We are grateful for
financial support from the European Commission (EU grants ICA4-
CT-2002-10051 and INCO-CT-2006-032321) and the VW foundation
(grant 1/81306). MB was the recipient of a travel grant by the Senior
Expert Service, Bonn, Germany.
We thank the study participants and the District
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
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