Doxycycline Treatment of Brugia malayi –Infected Persons Reduces Microfilaremia and Adverse...

Doxycycline Treatment in Brugian Filariasis • CID 2008:46 (1 May) • 1385

M A J O R A R T I C L E

Doxycycline Treatment of Brugia malayi–InfectedPersons Reduces Microfilaremia and AdverseReactions after Diethylcarbamazine and AlbendazoleTreatment

Taniawati Supali,1 Yenny Djuardi,1 Kenneth M. Pfarr,4 Heri Wibowo,1 Mark J. Taylor,5 Achim Hoerauf,4

Jeanine J. Houwing-Duistermaat,3 M. Yazdanbakhsh,2 and Erliyani Sartono2

1Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; Departments of 2Parasitology and 3MedicalStatistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands; 4Institute for Medical Microbiology, Immunology,and Parasitology, University of Bonn, Bonn, Germany; and 5Liverpool School of Tropical Medicine, Liverpool, United Kingdom

Background. The efficacy of doxycycline for treating the causal agent of human lymphatic filariasis, Brugiamalayi, is unknown. Standard treatment with diethylcarbamazine-albendazole is associated with adverse reactions.We assessed whether doxycycline alone or in combination with diethylcarbamazine-albendazole would lead tosustained amicrofilaremia and reduced incidence of adverse reactions.

Methods. A double-blind, randomized, placebo-controlled 6-week field trial of doxycycline treatment (100mg/day) of 161 persons infected with B. malayi was conducted. Four months after receiving doxycycline (n p

) or placebo ( ), participants received diethylcarbamazine (6 mg/kg) plus albendazole (400 mg) or a119 n p 42matching placebo. Adverse reactions were assessed 48 and 60 h after administration of diethylcarbamazine-alben-dazole. Treatment efficacy was evaluated at 2, 4, and 12 months after the initial doxycycline treatment.

Results. Four months after beginning doxycycline treatment, Wolbachia loads were reduced by 98%. Doxy-cycline treatment reduced the prevalence of microfilaremia at 2, 4, and 12 months of follow-up ( for allP ! .001time points). At the 1-year follow-up, prevalence was reduced by 77% and 87.5% in patients receiving doxycyclinealone or doxycycline plus diethylcarbamazine-albendazole, respectively. In contrast, the reduction of microfilaremiain the group receiving placebo doxycycline plus diethylcarbamazine-albendazole was merely 26.7%. Adverse re-actions were lowest in the group receiving doxycycline plus placebo diethylcarbamazine-albendazole and highestin the group receiving placebo doxycycline plus diethylcarbamazine-albendazole. The proportion of persons withhigh fever and severe adverse reactions was significantly reduced in the group treated with doxycycline plusdiethylcarbamazine-albendazole.

Conclusions. A 6-week course of doxycycline, either alone or in combination with diethylcarbamazine-alben-dazole, leads to a decrease in microfilaremia and reduces adverse reactions to antifilarial treatment in B. malayi–infected persons.

Brugia and Wuchereria species cause lymphatic filariasis,

which affects 120 million people worldwide [1]. Filarial

disease is associated with episodes of acute and chronic

inflammation that can lead to conditions such as ele-

phantiasis and hydrocele. The global lymphatic filariasis

Received 7 August 2007; accepted 23 December 2007; electronically published31 March 2008.

Reprints or correspondence: Dr. Taniawati Supali, Dept. of Parasitology, Facultyof Medicine, University of Indonesia, Salemba Raya 6, Jakarta, Indonesia([email protected]).

Clinical Infectious Diseases 2008; 46:1385–93� 2008 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2008/4609-0010$15.00DOI: 10.1086/586753

elimination program has 2 aims: (1) to reduce micro-

filaremia, by using filaricidal drugs, to levels that are

too low to sustain transmission of filarial parasites in

humans and (2) to reduce morbidity associated with

chronic filarial disease [1]. Current strategies involve

the community-based single oral treatment with com-

bination of diethylcarbamazine or ivermectin and al-

bendazole, which needs to be repeated annually or sem-

iannually over many years to reduce transmission [2,

3]. Additional control strategies are needed, because the

currently used drugs show only partial activity against

adult parasites [4].

The discovery of Wolbachia in filarial species has pro-

vided a new target for chemotherapy of human filariasis

1386 • CID 2008:46 (1 May) • Supali et al.

[5]. Wolbachia are susceptible to the tetracycline group of an-

tibiotics [6, 7]. Studies in animal models have shown that treat-

ment of infected animals with tetracycline decreases microfi-

larial load, inhibits development of larval worms, and renders

adult female worms infertile [8, 9]. In human onchocerciasis,

treatment with doxycycline (100 mg/day) for 6 weeks depleted

the bacteria and led to long-term sterility of adult female Onch-

ocerca volvulus [10]. Similarly, in bancroftian filariasis, a study

revealed that treatment of microfilaremic patients with doxy-

cycline (200 mg/day) led to a 96% reduction in Wolbachia DNA

levels. Twelve months after the commencement of doxycycline

therapy, there was an almost complete absence of microfilar-

emia [11, 12]. Recently, it was established that administration

of doxycycline (200 mg/day) for 8 weeks [13] or 6 weeks [14]

resulted in a strong macrofilaricidal activity.

Given the success of doxycycline treatment in depleting mi-

crofilariae in bancroftian filariasis as well as onchocerciasis, it

is essential to determine whether doxycycline in combination

with diethylcarbamazine-albendazole leads to sustained ami-

crofilaremia in brugian filariasis, because almost one-half of

persons affected by filariasis in Southeast Asia are infected with

Brugia malayi or Brugia timori [15]. Moreover, diethylcarbam-

azine treatment is poorly accepted in affected communities

because of adverse reactions of microfilaria-positive subjects to

this drug [16]. There is evidence that adverse reactions to di-

ethylcarbamazine coincide with the release of Wolbachia into

the bloodstream [17]. It is therefore important to assess the

effect of doxycycline on adverse reactions associated with di-

ethylcarbamazine-albendazole treatment. In the current study,

we aimed to assess the effect of targeting Wolbachia in brugian

filariasis as proof of principle that targeting the endosymbiont

of Brugia species is a valid strategy for discovering new ther-

apeutic candidates, with a high rate of community acceptance,

to be applied to an important, neglected disease.

MATERIALS AND METHODS

This European Union–funded collaborative study of European

and Indonesian institutions was approved by the Commission

of Medical Ethics of the University of Indonesia (Jakarta, In-

donesia) and for European centers by the Research Ethics Com-

mittee of the Liverpool School of Tropical Medicine (Liverpool,

United Kingdom). The trial registration number is ISRTN

37962059.

Objectives. The primary objective was to assess whether a

6-week course of doxycycline treatment was effective in re-

ducing Wolbachia loads in B. malayi–infected persons and,

when given in combination with diethylcarbamazine plus al-

bendazole, whether treatment would result in sustained ami-

crofilaremia in subjects with brugian filariasis. The secondary

objective was to reduce adverse reactions to standard antifilarial

drugs.

Study site and enrollment. The study was performed from

March 2002 through August 2003 in Parigi-Moutong, Central

Sulawesi, and in Bonebolanggo, Northern Sulawesi, areas of

Indonesia where B. malayi is endemic. Before the trial, the

prevalences of microfilariae in the villages in Parigi-Moutong

and Bonebolanggo were 24% and 38%, respectively. The in-

habitants were informed of the purpose of the study. Informed

consent was obtained from all persons (or parents) before the

clinical and parasitological study and blood withdrawal in ac-

cordance with the guidelines of Indonesian Department of

Health and Human Services. Eligibility requirements for par-

ticipation were as follows: age range, 12–76 years; body weight,

�40 kg; microfilaria load, 15 microfilaria/mL; and good health

without any clinical condition requiring long-term use of

medications.

The study included 161 participants with microfilaremia. The

median age for men was 29 years (range, 12–76 years) and for

women was 28.5 years (range, 12–70 years). Exclusion criteria

for all participants were abnormal hepatic and renal profiles

(alanine aminotransferase level, 130 U/L; g-glutamyl transpep-

tidase level, 128 U/L; creatinine level, 11.2 mg/dL) measured

by dipstick chemistry (Reflotron; Roche Diagnostics), and ad-

ditional exclusion criteria for women included pregnancy or

breast-feeding.

Interventions and randomization. Doxycycline (100 mg)

and matched placebo capsules were provided by Pfizer. Dieth-

ylcarbamazine, albendazole, and placebo tablets were purchased

from Indo Farma Pharmaceutical Company.

To prevent mix-up of the drugs and matching placebos

within members of the same family, the participants were as-

signed to groups by cluster randomization. The drugs and pla-

cebo capsules were coded with random numbers, which were

concealed from investigators and patients. Groups I and II

received 1 capsule of doxycyline (100 mg) per day for 6 weeks;

group III received 1 capsule of matching placebo per day for

6 weeks (figure 1). Drugs were taken under supervision of the

medical team, and adverse reactions were monitored and re-

corded in a questionnaire completed by medical staff. During

these 6-week courses of doxycycline treatment, the patients

were visited by primary health care medical staff who were

unaware of treatment allocation daily.

Four months after the start of treatment, groups II and III

received a single dose of diethylcarbamazine (6 mg/kg) plus

albendazole (400 mg), whereas group I received placebo instead

of diethylcarbamazine and albendazole. For diethylcarbama-

zine-albendazole and placebo diethylcarbamazine-albendazole

treatment, all patients were admitted to a primary health center

for 3 days, which allowed adverse reactions to be monitored

closely and frequently (twice daily) and for appropriate care to

be provided to the patients. Tablets were swallowed under su-

pervision after a meal. Heart rate, blood pressure, and body

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Table 1. Microfilaraemia before and after treatment with doxycycline (DOX) and/or diethylcarbamazine-albendazole (DEC).

Group, parameter

Time relative to treatments

Before DOX2 monthsafter DOX

4 months after DOX(before DEC)a

3 daysafter DEC

7 daysafter DEC

1 yearafter DOXa

DOX plus placebo DEC

No. of patients (no. female/no. male) 54 (27/27) 53 (26/27) 54 (27/27) 54 (27/27) 54 (27/27) 48 (26/22)

Microfilaria, geometric mean count (95% CI) 328 (225–477) 271 (159–459) 37 (20–459)b 27 (15–49)b 24 (13–43)b 2 (1–2)b

Proportion of patients with microfilaremia (%) 54/54 (100) 51/53 (96.2) 43/54 (79.6) 41/54 (75.9) 42/54 (77.8) 11/48 (22.9)

DOX plus DEC


Microfilaria, geometric mean count (95% CI) 506 (373–687) 455 (256–808)c 89 (51–158)b 12 (8–19)b 13 (8–19)b 1 (1–1)b

Proportion of patients with microfilaremia (%) 42/42 (100) 37/41 (90.2) 39/42 (92.9) 36/42 (85.7) 35/42 (83.3) 5/40 (12.5)

Placebo DOX plus DEC


Microfilaria, geometric mean count (95% CI) 373 (247–562) 403 (246–660)d 227 (140–369)d 20 (11–30)b 39 (23–65)b 13 (6–30)b

Proportion of patients with microfilaremia (%) 42/42 (100) 37/37 (100) 42/42 (100) 38/42 (90.5) 39/39 (100) 22/30 (73.3)

a for comparison of microfilaria count between treatment groups using Kruskal-Wallis nonparametric analyses of variance.P ! .001b , compared with predoxycycline value (Wilcoxon signed rank test).P ! .001c , compared with predoxycycline value (Wilcoxon signed rank test).P ! .05d , compared with predoxycycline value (Wilcoxon signed rank test).P ! .01

temperature (measured at the armpit for 15 min) were mea-

sured. Any clinical findings and complaints were recorded.

The calculation of sample size was based on the mean mi-

crofilaria density in a pilot study, reduction in microfilaria den-

sity by 50% after 4 months (power, 80%; a p 0.05), and a

15% dropout rate throughout the trial period. For assessment

of the potential reduction in adverse reactions to treatment

with diethylcarbamazine-albendazole, a group size of 13 per-

sons per treatment allocation was derived from power calcu-

lations based on a pilot study, assuming that 50% of persons

in the control group have elevated proinflammatory cytokine

levels, compared with none of the doxycycline-treated persons

(power, 80%; a p 0.05).

Outcomes. The primary outcome measurements were the

quantity of Wolbachia single-copy genes per microfilaria (as a

surrogate for numbers of whole bacteria per microfilaria) 4

months after the start of treatment and the quantity of cir-

culating microfilariae at 2, 4, and 12 months after the start of

treatment. The numbers of Wolbachia per microfilaria and the

microfilaria level were determined by collecting 5 mL of night

blood (obtained between 8:00 and 10:00 p.m.) in an EDTA-

containing tube. One mL of blood was filtered through a 5-

mm–pore filter (Millipore) and stained with Giemsa to quantify

microfilariae. The filtration and counting of the microfilariae

was performed by the same person on all occasions. The plasma

from the rest of the blood was collected by centrifugation and

stored at �20�C for serological assays. The remaining blood

pellet was diluted with 5 mL of distilled water and filtered to

retain microfilariae for quantification of Wolbachia DNA.

Wolbachia content was quantified before doxycycline treat-

ment and at 2 and 4 months after doxycyline treatment. Ge-

nomic DNA was extracted from microfilariae collected from

blood specimens on filters with the QIAamp DNA kit (Qiagen).

The single-copy ftsZ gene of Wolbachia was quantified by real-

time PCR, using 1� HotStar Taq polymerase buffer (Qiagen),

3.75 mmol/L MgCl2, 200 mmol/L dNTPs, 300 nmol/L each of

forward (5′-CGATGAGATTATGGAGCATATAA-3′) and re-

verse (5′-TTGCAATTACTGGTGCTGC-3′) primers, 50 nmol/

L hybridization probe (5′-[Fam]-CAGGGATGGGTGGTGGC-

ACTGGAA-BHQ1-3′), 2.5 U of HotStar Taq (Qiagen), and 2

mL of DNA in a 20-mL total reaction volume. Amplification

took place in a Rotorgene 3000 (Corbett Research) under the

following conditions: 1 cycle of 15 min at 95�C, followed by

40 cycles of 94�C for 15 s, 58�C for 30 s, and 72�C for 30 s,

with fluorescence monitored on the FAM channel. Copy num-

bers of ftsZ were calculated from a standard curve of a serially

diluted plasmid containing ftsZ and then divided by microfi-

lariae per microliter.

Blood samples were collected at the following time points:

before doxycycline treatment; 2 months after the start of dox-

ycycline treatment; 4 months after the start of doxycycline treat-

ment or just before diethylcarbamazine-albendazole treatment;

3 days and 7 days after diethylcarbamazine-albendazole treat-

ment; and 1 year after the start of doxycycline treatment.

The secondary outcome measurement was the clinical as-

sessment of adverse reactions following antifilarial treatment.

Symptoms were monitored during the 3 days after adminis-

tration of diethylcarbamazine-albendazole. The scoring of

symptoms was as reported elsewhere [18]. Briefly, 0 points were

awarded for the absence of symptoms and a body temperature

�37.4�C; 1 point each for the presence of arthralgia, dizziness,

lymph node enlargement, or myalgia; and 2 points for a head-

ache. Body temperatures measuring 37.5�–38.5�C were awarded

5 points, and temperatures 138.5�C were awarded 10 points.


Figure 2. Microfilaria (Mf) count in 1 mL of night blood before dox-ycycline treatment (pre-DOX), 4 months after the beginning of doxycyclinetreatment before administration of diethylcarbamazine-albendazole treat-ment (pre-DEC), 7 days after diethylcarbamazine-albendazole (7d-DEC),and 1 year after the onset of doxycycline treatment (1yr-DOX) in groupstreated with doxycycline plus placebo diethylcarbamazine-albendazole(DOX & pl DEC), doxycycline plus diethylcarbamazine-albendazole (DOX& DEC), and placebo doxycycline plus diethylcarbamazine-albendazole (plDOX & DEC). Each mark represents 1 person; horizontal lines representmedians. *P value from nonparametric repeated-measures trend test.** for comparison of changes in microfilaria count with baselineP ! .001predoxycycline levels, determined using the Wilcoxon signed rank test.

Adverse reactions were classified into 3 categories exactly as

described elsewhere [18].

Statistical analysis. Statistical analysis was performed in

SPSS for Windows, version 11 (SPSS). Differences between var-

iables for 3 groups were assessed by use of the Kruskal-Wallis

test, whereas the Mann-Whitney U test was used for comparing

differences between 2 groups. A nonparametric, repeated-mea-

sure trend test was performed to assess the effect of treatment

over time. Specifically, measurements were ranked within in-

dividuals, followed by a repeated-measurement trend test. Dif-

ferences in the variables before and after treatment of the same

individual were compared using the Wilcoxon matched-pairs

rank test. Comparison of proportions was performed using the

x2 test.

RESULTS

Adverse reactions to doxycycline and diethylcarbamazine-

albendazole. The 6-week course of 100 mg of doxycyline per

day was generally well tolerated. The adverse reactions reported

after doxycycline administration were mild and tolerable. None

of the participants had to stop taking doxycycline because of

adverse reactions. The adverse effects in the doxycycline group

( ) were myalgia ( ), nausea ( ), dizzinessn p 119 n p 6 n p 4

( ), headache ( ), insomnia ( ), abdominal painn p 5 n p 3 n p 2

( ), itching ( ), diarrhea ( ), rash ( ), mal-n p 2 n p 2 n p 1 n p 1

aise ( ), and drowsiness ( ); in the placebo groupn p 1 n p 1

( ), adverse effects were myalgia ( ), nausea (n p 42 n p 2 n p

), dizziness ( ), insomnia ( ), and itching ( ).1 n p 2 n p 1 n p 1

No report of photosensitivity reactions was recorded during or

after the treatment.

The most common adverse reactions to diethylcarbamazine-

albendazole were fever, headache, arthralgia, dizziness, lymph

node enlargement, or myalgia. Altogether, 51 of 99 participants

receiving diethylcarbamazine-albendazole and 8 of 62 receiving

placebo diethylcarbamazine-albendazole experienced moderate

or severe adverse reactions.

Reduction of microfilaria count and prevalence of micro-

filaremia after doxycycline and diethylcarbamazine-alben-

dazole treatment. The microfilaria count before doxycycline

treatment was the same in all 3 treatment groups. Four months

after the start of doxycycline treatment, the microfilaria count

in each group was reduced significantly compared with that

before doxycycline treatment ( for all groups). The re-P ! .001

duction in microfilaria count was highest in the groups treated

with doxycycline and lowest in the group treated with placebo

doxycycline (89%, 83%, and 39% for the doxycycline plus pla-

cebo diethylcarbamazine-albendazole group, the doxycycline

plus diethylcarbamazine-albendazole group, and the placebo

doxycycline plus diethylcarbamazine-albendazole group, re-

spectively). During diethylcarbamazine-albendazole treatment,

there was a strong reduction in microfilaria counts in both


Table 2. Effect of doxycycline treatment on Wolbachia ftsZ gene copies per microfilaria.

Treatment group, time (months)Proportion ofpatients (%)

ftsZ gene,median copies per microfilaria

(10th–90th percentile) P a

Doxycycline plus placebo diethylcarbamazine-albendazoleb

0 60/60 (100) 204 (14–1278)2 58/61 (95) 12 (3–73) !.0014 40/61 (66) 5 (1–60) !.001

Doxycycline plus diethylcarbamazine-albendazoleb

0 55/55 (100) 226 (64–1874)2 52/56 (93) 13 (3–102) !.0014 42/57 (74) 5 (1–437) !.001

Placebo doxycycline plus diethylcarbamazine-albendazolec

0 39/39 (100) 470 (41–1723)2 35/35 (100) 599 (79–1420) .44 41/41 (100) 312 (21–823) .02

a Changes from baseline in Wolbachia ftsZ gene copy numbers per microfilaria.b , assessed by nonparametric repeated measure trend test.P ! .001c , assessed by nonparametric repeated measure trend test.P p .28

placebo doxycycline and doxycycline groups, and at 7 days after

the administration of diethylcarbamazine-albendazole, the lev-

els of microfilaria in those 2 groups were comparable. However,

after 1 year, the group receiving doxycycline plus diethylcar-

bamazine-albendazole had significantly reduced levels of mi-

crofilaria, compared with placebo doxycycline plus diethylcar-

bamazine-albendazole recipients.

Interestingly, at 1 year after doxycycline treatment the dox-

ycycline plus placebo diethylcarbamazine-albendazole group

had a significantly lower microfilaria count than did the group

receiving placebo doxycycline plus diethylcarbamazine-alben-

dazole (table 1 and figure 2). The reduction in prevalence of

microfilaremia 1 year after doxycycline treatment was 77% and

87.5% in the doxycycline plus placebo diethylcarbamazine-al-

bendazole and the doxycycline plus diethylcarbamazine-alben-

dazole groups, respectively; in contrast, the reduction in the

placebo doxycycline plus diethylcarbamazine-albendazole

group was only 26.7% (table 1). One year after initiation of

doxycycline or placebo doxycycline treatment, both doxycycline

plus placebo diethylcarbamazine-albendazole and doxycycline

plus diethylcarbamazine-albendazole groups had lower num-

bers of microfilaria-positive participants and lower microfilaria

density than did the placebo doxycycline plus diethylcarbam-

azine-albendazole group. The prevalence of microfilaremia as

well as the microfilaria count in the doxycycline plus diethyl-

carbamazine-albendazole group was lower than in the doxy-

cycline plus placebo diethylcarbamazine-albendazole group,

but this did not reach statistical significance ( for bothP p .09

prevalence of microfilaremia and microfilaria count).

Reduction of Wolbachia levels after doxycyline treatment.

DNA samples from microfilariae trapped on filters were ana-

lyzed to determine Wolbachia ftsZ gene copy numbers per mi-

crofilaria. There were no differences in the gene copy numbers

between the 3 treatment groups at the start of the trial.

The gene copy numbers of microfilaria-positive, ftsZ-positive

participants were reduced by 94% in both doxycycline groups

at 2 months ( for both groups), compared with beforeP ! .001

doxycycline treatment. At this time point, proportions of pa-

tients with no microfilariae and no ftsZ copies were 5% and

7% in the doxycycline plus placebo diethylcarbamazine-alben-

dazole and doxycycline plus diethylcarbamazine-albendazole

groups, respectively.

At 4 months after the onset of treatment, ftsZ copy numbers

were reduced by 98% in both doxycycline groups compared

with levels before doxycycline treatment. At this time point,

we also found a slight reduction in ftsZ copy numbers in the

placebo doxycycline group ( ), compared with beforeP ! .05

doxycycline treatment (table 2).

Body temperature and adverse reactions after diethylcar-

bamazine-albendazole treatment. Body temperatures and

adverse reaction scores were lowest in the group that received

doxycycline and placebo diethylcarbamazine-albendazole. In

the doxycycline plus placebo diethylcarbamazine-albendazole

group, no one had severe adverse reactions or a body temper-

ature 138�C. The effect of doxycycline on adverse reactions to

diethylcarbamazine-albendazole was clear from the significantly

lower body temperatures and adverse reactions in doxycycline

plus diethylcarbamazine-albendazole recipients, compared with

those in placebo doxycycline plus diethylcarbamazine-alben-

dazole recipients (figure 3). The percentage of persons with

body temperature 138�C or severe adverse reactions was sig-

nificantly higher in the placebo doxycycline plus diethylcar-

bamazine-albendazole group (26.3% and 42.9%, respectively),

compared with the doxycycline plus diethylcarbamazine-alben-


Figure 3. Body temperature (A) and adverse reactions (B) followingdiethylcarbamazine-albendazole treatment in groups treated with doxy-cycline plus placebo diethylcarbamazine-albendazole (DOX & pl DEC),doxycycline plus diethylcarbamazine-albendazole (DOX & DEC), and pla-cebo doxycycline plus diethylcarbamazine-albendazole (pl DOX & DEC).Each mark represents 1 person; horizontal lines represent medians. *Dif-ferences between body temperature and adverse reactions in treatmentgroups were assessed using Kruskal-Wallis nonparametric analysis ofvariance.

dazole group (10.5% and 15.8%, respectively; ) (ta-P ! .05

ble 3).

DISCUSSION

Doxycycline treatment depleted Wolbachia from B. malayi in

infected patients; this is in agreement with studies of bancrof-

tian filariasis [11–13]. Levels of Wolbachia measured before

doxycycline treatment, as determined by copy numbers of the

ftsZ gene per microfilaria (median, 300 copies/microfilaria),

agree well with copy numbers determined for the wsp gene in

a laboratory strain of B. malayi [19]. After doxycycline treat-

ment, the Wolbachia load per microfilaria was reduced by 11

log, a 94% reduction compared with pre-doxycycline levels.

Four months after treatment, the Wolbachia load was reduced

by 2 logs, representing a 98% reduction compared with pre-

doxycycline levels.

Our results have established that a 6-week course of 100 mg

doxycycline per day leads to a strong reduction in microfilar-

emia in B. malayi–infected subjects. Moreover, diethylcarbam-

azine-albendazole without doxycycline induced a significant re-

duction in microfilaremia; however, only 27% of subjects were

amicrofilaremic 1 year after treatment, compared with much

higher rates of amicrofilaremia in the doxycycline plus dieth-

ylcarbamazine-albendazole (87.5%) or the doxycycline plus

placebo diethylcarbamazine-albendazole (77%) groups. Here,

we have also shown that microfilaria count as well as prevalence

of microfilaremia at 1 year after treatment was similar in par-

ticipants receiving doxycycline plus diethylcarbamazine-alben-

dazole and in those receiving doxycycline plus placebo dieth-

ylcarbamazine-albendazole, indicating that doxycycline by itself

was highly effective in reducing B. malayi levels. Together, these

results imply that a 6-week course of 100-mg/day doxycycline

alone is sufficient to deplete Wolbachia from filarial worms and

to lead to almost complete amicrofilaremia at 1 year after treat-

ment. Although the measurement of adult worm burden is not

possible for brugian filariasis (unlike bancroftian filariasis) [20,

21], the fact that doxycycline leads to a strong long-term (12-

month) reduction in microfilaremia (in contrast to 1 dose of

diethylcarbamazine-albendazole) suggests that doxycycline ei-

ther leads to long-term sterilization of adult female Brugia or

has substantial macrofilaricidal effects. A strong macrofilaricidal

effect has been shown in humans infected with W. bancrofti

[13, 14] as well as in gerbils infected with Brugia pahangi

[8, 9].

We also observed a small but significant reduction in mi-

crofilaria counts at 4 months in the placebo doxycycline group,

but the microfilaremia prevalence remained at 100%. The levels

of microfilariae have been shown to fluctuate seasonally [22].

Pretreatment samples had been collected at the end of the rainy

season, whereas the samples before diethylcarbamazine-alben-

dazole treatment were collected in the dry season; thus, sea-

sonality may account for the slight reduction in the microfilarial

density at 4 months after treatment in the placebo doxycycline

group. The seasonal fluctuation may also account for the sig-

nificant reduction in the Wolbachia load seen in the placebo

doxycycline group 4 months after the start of treatment.

Diethylcarbamazine treatment is known to induce local and

systemic adverse reactions [18]. The adverse reactions are

thought to be caused by the rapid release of microfilarial ma-

terial and Wolbachia into the bloodstream [3, 17]. Because body

temperature is a continuous variable and a major adverse re-

action to diethylcarbamazine, we also analyzed it separately. In

contrast to the placebo diethylcarbamazine-albendazole group,


Table 3. Adverse reactions and elevations in body temperature in groups treated with doxycycline or placebo plus dieth-ylcarbamazine-albendazole or placebo.

Measure

Data are proportion (%) of patients

Doxycyclineplus placebo

diethylcarbamazine-albendazole

Doxycyclineplus


Placebo doxycyclineplus


Body temperature category!37.5�C 60/62 (96.8) 39/57 (68.4) 19/42 (45.2)37.5�C–38.5�C 2/62 (3.2) 12/57 (21.1) 12/42 (28.6)138.5�C 0/62 (0)a 6/57 (10.5)b 11/42 (26.2)

Adverse reaction scoreMild 54/62 (87.1) 33/57 (57.9) 15/42 (35.7)Moderate 8/62 (12.9) 15/57 (26.3) 9/42 (21.4)Severe 0/62 (0)a 9/57 (15.8)c 18/42 (42.9)

a for comparison of doxycycline plus placebo diethylcarbamazine-albendazole and doxycycline plus diethylcarbamazine-albendazole groups,P ! .001by x2 test.

b for comparison of doxycycline plus diethylcarbamazine-albendazole and placebo doxycycline plus diethylcarbamazine-albendazole groups,P ! .05by x2 test.

c for comparison of doxycycline plus diethylcarbamazine-albendazole and placebo doxycycline plus diethylcarbamazine-albendazole groups,P ! .01by x2 test.

we observed higher body temperatures or adverse reactions in

groups treated with diethylcarbamazine-albendazole. When the

2 groups treated with diethylcarbamazine-albendazole were

compared, we found ∼1.5 fold higher percentage of persons

with body temperatures !37.5�C or mild adverse reactions in

the group treated with doxycycline before diethylcarbamazine-

albendazole than in the group who did not receive doxycycline.

Moreover, we found that the percentages of persons experi-

encing body temperatures 138�C or severe adverse reactions

were 2 to 3 times lower when doxycycline was received prior

to diethylcarbamazine-albendazole treatment, suggesting that

anti-Wolbachia treatment may prevent adverse effects of anti-

filarial treatment.

In conclusion, findings reported here support the notion that

doxycycline, either alone or in combination with antifilarial

therapy, strongly reduced Wolbachia levels in B. malayi–infected

persons and, either alone or in combination with antifilarial

therapy, led to a decrease in microfilaremia that was sustained

for at least 1 year after treatment. Second, doxycycline treatment

reduced adverse reactions following diethylcarbamazine-alben-

dazole treatment; this may be particularly important for pa-

tients with very high microfilaria loads, because they are known

to most frequently experience adverse reactions to

diethylcarbamazine.

The findings are important at 2 levels. At the individual level,

doxycycline might be a suitable alternative for individual treat-

ment and for those wishing to avoid adverse reactions to stan-

dard treatments. At the community level, although doxycycline

is not appropriate for mass drug administration, our findings

provide proof of principle that the endosymbiont of Brugia

species is a valid target for new drug discovery and development

[23, 24].

Acknowledgments

We gratefully acknowledge the authority district staffs at Palu and Go-rontalo for their encouragement and help during the trial. We thank Pfizerfor providing drug treatment free of charge.

Financial support. The European Union (European Commission,INCO programme, contract no. IC4-CT-2002-10051).

Potential conflicts of interest. All authors: no conflicts.

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Doxycycline Treatment of Brugia malayi –Infected Persons Reduces Microfilaremia and Adverse...

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