MAJOR ARTICLE Doxycycline Treatment of Brugia malayi Infected Persons Reduces Microfila emia and Adverse Reactions after Diethylcarbamazine and Albendazole Treatment 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 Sartono 2 1 Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; Departments of 2 Parasitology and 3 Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands; 4 Institute for Medical Microbiology, Immunology, and Parasitology, University of Bonn, Bonn, Germany; and 5 Liverpool School of Tropical Medicine, Liverpool, United Kingdom Background. The efficac of doxycycline for treating the causal agent of human lymphatic filariasis Brugia malayi, 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 to sustained amicrofila emia and reduced incidence of adverse reactions. Methods. A double-blind, randomized, placebo-controlled 6-week fiel trial of doxycycline treatment (100 mg/day) of 161 persons infected with B. malayi was conducted. Four months after receiving doxycycline (n p 119) or placebo ( n p 42), participants received diethylcarbamazine (6 mg/kg) plus albendazole (400 mg) or a matching placebo. Adverse reactions were assessed 48 and 60 h after administration of diethylcarbamazine-albendazole. Treatment efficac 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%. Doxycycline treatment reduced the prevalence of microfila emia at 2, 4, and 12 months of follow-up ( P!.001 for all time points). At the 1-year follow-up, prevalence was reduced by 77% and 87.5% in patients receiving doxycycline alone or doxycycline plus diethylcarbamazine-albendazole, respectively. In contrast, the reduction of microfila emia in the group receiving placebo doxycycline plus diethylcarbamazine-albendazole was merely 26.7%. Adverse reactions were lowest in the group receiving doxycycline plus placebo diethylcarbamazine-albendazole and highest in the group receiving placebo doxycycline plus diethylcarbamazine-albendazole. The proportion of persons with high fever and severe adverse reactions was significantl reduced in the group treated with doxycycline plus diethylcarbamazine-albendazole. Conclusions. A 6-week course of doxycycline, either alone or in combination with diethylcarbamazine-albendazole, leads to a decrease in microfila emia and reduces adverse reactions to antifilaria 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 inflammatio that can lead to conditions such as elephantiasis and hydrocele. The global lymphatic filariasi Received 7 August 2007; accepted 23 December 2007; electronically published 31 March 2008. Reprints or correspondence: Dr. Taniawati Supali, Dept. of Parasitology, Faculty of Medicine, University of Indonesia, Salemba Raya 6, Jakarta, Indonesia (taniawati@yahoo.com). Clinical Infectious Diseases 2008; 46:1385 93 2008 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2008/4609-0010$15.00 DOI: 10.1086/586753 elimination program has 2 aims: (1) to reduce microfila emia, by using filaricida drugs, to levels that are too low to sustain transmission of filaria parasites in humans and (2) to reduce morbidity associated with chronic filaria disease [1]. Current strategies involve the community-based single oral treatment with combination of diethylcarbamazine or ivermectin and albendazole, which needs to be repeated annually or semiannually 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 filaria species has provided a new target for chemotherapy of human filariasi Doxycycline Treatment in Brugian Filariasis CID 2008:46 (1 May) 1385
[5]. Wolbachia are susceptible to the tetracycline group of antibiotics [6, 7]. Studies in animal models have shown that treatment 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 Onchocerca volvulus [10]. Similarly, in bancroftian filariasis a study revealed that treatment of microfila emic patients with doxycycline (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 microfila - 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 macrofilaricida activity. Given the success of doxycycline treatment in depleting microfilaria in bancroftian filariasi as well as onchocerciasis, it is essential to determine whether doxycycline in combination with diethylcarbamazine-albendazole leads to sustained amicrofila emia in brugian filariasis because almost one-half of persons affected by filariasi in Southeast Asia are infected with Brugia malayi or Brugia timori [15]. Moreover, diethylcarbamazine treatment is poorly accepted in affected communities because of adverse reactions of microfilaria-positiv subjects to this drug [16]. There is evidence that adverse reactions to diethylcarbamazine 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 diethylcarbamazine-albendazole treatment. In the current study, we aimed to assess the effect of targeting Wolbachia in brugian filariasi as proof of principle that targeting the endosymbiont of Brugia species is a valid strategy for discovering new therapeutic 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, Indonesia) and for European centers by the Research Ethics Committee 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 reducing Wolbachia loads in B. malayi infected persons and, when given in combination with diethylcarbamazine plus albendazole, whether treatment would result in sustained amicrofila emia in subjects with brugian filariasis The secondary objective was to reduce adverse reactions to standard antifilaria 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 microfilaria in the villages in Parigi-Moutong and Bonebolanggo were 24% and 38%, respectively. The inhabitants 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 accordance with the guidelines of Indonesian Department of Health and Human Services. Eligibility requirements for participation were as follows: age range, 12 76 years; body weight, 40 kg; microfilari load, 15 microfilaria/ml and good health without any clinical condition requiring long-term use of medications. The study included 161 participants with microfila emia. 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 profile (alanine aminotransferase level, 130 U/L; g-glutamyl transpeptidase level, 128 U/L; creatinine level, 11.2 mg/dl) measured by dipstick chemistry (Reflot on; Roche Diagnostics), and additional exclusion criteria for women included pregnancy or breast-feeding. Interventions and randomization. Doxycycline (100 mg) and matched placebo capsules were provided by Pfize. Diethylcarbamazine, 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 assigned to groups by cluster randomization. The drugs and placebo 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 (figu e 1). Drugs were taken under supervision of the medical team, and adverse reactions were monitored and recorded 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 diethylcarbamazine-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 supervision after a meal. Heart rate, blood pressure, and body 1386 CID 2008:46 (1 May) Supali et al.
Figure 1. Design of a study of doxycycline treatment plus treatment with diethylcarbamazine-albendazole among persons infected with Brugia malayi.
Table 1. Microfilaraemia before and after treatment with doxycycline (DOX) and/or diethylcarbamazine-albendazole (DEC). Group, parameter DOX plus placebo DEC Before DOX 2 months after DOX Time relative to treatments 4 months after DOX (before DEC) a 3 days after DEC 7 days after DEC 1 year after DOX a 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 No. of patients (no. female/no. male) 57 (21/36) 56 (21/35) 57 (21/36) 57 (21/36) 56 (21/35) 54 (21/33) 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 No. of patients (no. female/no. male) 42 (21/21) 37 (20/17) 42 (21/21) 42 (21/21) 39 (19/20) 30 (13/17) 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 P!.001 b P!.001, compared with predoxycycline value (Wilcoxon signed rank test). c P!.05, compared with predoxycycline value (Wilcoxon signed rank test). d P!.01, compared with predoxycycline value (Wilcoxon signed rank test). for comparison of microfilaria count between treatment groups using Kruskal-Wallis nonparametric analyses of variance. temperature (measured at the armpit for 15 min) were measured. Any clinical finding and complaints were recorded. The calculation of sample size was based on the mean microfilari density in a pilot study, reduction in microfilari density 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 persons per treatment allocation was derived from power calculations based on a pilot study, assuming that 50% of persons in the control group have elevated proinflammato y 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 microfilari (as a surrogate for numbers of whole bacteria per microfilaria 4 months after the start of treatment and the quantity of circulating microfilaria at 2, 4, and 12 months after the start of treatment. The numbers of Wolbachia per microfilari and the microfilari level were determined by collecting 5 ml of night blood (obtained between 8:00 and 10:00 p.m.) in an EDTAcontaining tube. One ml of blood was filte ed through a 5- mm pore filte (Millipore) and stained with Giemsa to quantify microfilariae The filtratio and counting of the microfilaria 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 filte ed to retain microfilaria for quantificatio of Wolbachia DNA. Wolbachia content was quantifie before doxycycline treatment and at 2 and 4 months after doxycyline treatment. Genomic DNA was extracted from microfilaria collected from blood specimens on filter with the QIAamp DNA kit (Qiagen). The single-copy ftsz gene of Wolbachia was quantifie by realtime PCR, using 1 HotStar Taq polymerase buffer (Qiagen), 3.75 mmol/l MgCl 2, 200 mmol/l dntps, 300 nmol/l each of forward (5 -CGATGAGATTATGGAGCATATAA-3 ) and reverse (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. Amplificatio 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 fluo escence monitored on the FAM channel. Copy numbers 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 doxycycline treatment; 4 months after the start of doxycycline treatment or just before diethylcarbamazine-albendazole treatment; 3 days and 7 days after diethylcarbamazine-albendazole treatment; and 1 year after the start of doxycycline treatment. The secondary outcome measurement was the clinical assessment of adverse reactions following antifilaria treatment. Symptoms were monitored during the 3 days after administration of diethylcarbamazine-albendazole. The scoring of symptoms was as reported elsewhere [18]. Briefl, 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 headache. Body temperatures measuring 37.5 38.5 C were awarded 5 points, and temperatures 138.5 C were awarded 10 points. 1388 CID 2008:46 (1 May) Supali et al.
Adverse reactions were classifie into 3 categories exactly as described elsewhere [18]. Statistical analysis. Statistical analysis was performed in SPSS for Windows, version 11 (SPSS). Differences between variables 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-measure trend test was performed to assess the effect of treatment over time. Specificall, measurements were ranked within individuals, followed by a repeated-measurement trend test. Differences 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 x 2 test. RESULTS Figure 2. Microfilaria (Mf) count in 1 ml of night blood before doxycycline treatment (pre-dox), 4 months after the beginning of doxycycline treatment before administration of diethylcarbamazine-albendazole treatment (pre-dec), 7 days after diethylcarbamazine-albendazole (7d-DEC), and 1 year after the onset of doxycycline treatment (1yr-DOX) in groups treated with doxycycline plus placebo diethylcarbamazine-albendazole (DOX & pl DEC), doxycycline plus diethylcarbamazine-albendazole (DOX & DEC), and placebo doxycycline plus diethylcarbamazine-albendazole (pl DOX & DEC). Each mark represents 1 person; horizontal lines represent medians. *P value from nonparametric repeated-measures trend test. ** P!.001 for comparison of changes in microfilaria count with baseline predoxycycline levels, determined using the Wilcoxon signed rank test. Adverse reactions to doxycycline and diethylcarbamazinealbendazole. 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 ( n p 119) were myalgia ( n p 6), nausea ( n p 4), dizziness ( n p 5), headache ( n p 3), insomnia ( n p 2), abdominal pain ( n p 2), itching ( n p 2), diarrhea ( n p 1), rash ( n p 1), mal- aise ( n p 1), and drowsiness ( n p 1); in the placebo group ( n p 42), adverse effects were myalgia ( n p 2), nausea ( n p 1), dizziness ( n p 2), insomnia ( n p 1), and itching ( n p 1). No report of photosensitivity reactions was recorded during or after the treatment. The most common adverse reactions to diethylcarbamazinealbendazole 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 microfilari count and prevalence of microfila emia after doxycycline and diethylcarbamazine-albendazole treatment. The microfilari count before doxycycline treatment was the same in all 3 treatment groups. Four months after the start of doxycycline treatment, the microfilari count in each group was reduced significantl compared with that before doxycycline treatment ( P!.001 for all groups). The reduction in microfilari 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 placebo diethylcarbamazine-albendazole group, the doxycycline plus diethylcarbamazine-albendazole group, and the placebo doxycycline plus diethylcarbamazine-albendazole group, respectively). During diethylcarbamazine-albendazole treatment, there was a strong reduction in microfilari counts in both Doxycycline Treatment in Brugian Filariasis CID 2008:46 (1 May) 1389
Table 2. Effect of doxycycline treatment on Wolbachia ftsz gene copies per microfilaria. Treatment group, time (months) Doxycycline plus placebo diethylcarbamazine-albendazole b Proportion of patients (%) ftsz gene, median copies per microfilaria (10th 90th percentile) 0 60/60 (100) 204 (14 1278) 2 58/61 (95) 12 (3 73)!.001 4 40/61 (66) 5 (1 60)!.001 Doxycycline plus diethylcarbamazine-albendazole b 0 55/55 (100) 226 (64 1874) 2 52/56 (93) 13 (3 102)!.001 4 42/57 (74) 5 (1 437)!.001 Placebo doxycycline plus diethylcarbamazine-albendazole c 0 39/39 (100) 470 (41 1723) 2 35/35 (100) 599 (79 1420).4 4 41/41 (100) 312 (21 823).02 a Changes from baseline in Wolbachia ftsz gene copy numbers per microfilaria. b P!.001, assessed by nonparametric repeated measure trend test. c P p.28, assessed by nonparametric repeated measure trend test. P a placebo doxycycline and doxycycline groups, and at 7 days after the administration of diethylcarbamazine-albendazole, the levels of microfilari in those 2 groups were comparable. However, after 1 year, the group receiving doxycycline plus diethylcarbamazine-albendazole had significantl reduced levels of microfilaria compared with placebo doxycycline plus diethylcarbamazine-albendazole recipients. Interestingly, at 1 year after doxycycline treatment the doxycycline plus placebo diethylcarbamazine-albendazole group had a significantl lower microfilari count than did the group receiving placebo doxycycline plus diethylcarbamazine-albendazole (table 1 and figu e 2). The reduction in prevalence of microfila emia 1 year after doxycycline treatment was 77% and 87.5% in the doxycycline plus placebo diethylcarbamazine-albendazole and the doxycycline plus diethylcarbamazine-albendazole 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 numbers of microfilaria-positiv participants and lower microfilari density than did the placebo doxycycline plus diethylcarbamazine-albendazole group. The prevalence of microfila emia as well as the microfilari count in the doxycycline plus diethylcarbamazine-albendazole group was lower than in the doxycycline plus placebo diethylcarbamazine-albendazole group, but this did not reach statistical significanc ( P p.09 for both prevalence of microfila emia and microfilari count). Reduction of Wolbachia levels after doxycyline treatment. DNA samples from microfilaria trapped on filter were analyzed to determine Wolbachia ftsz gene copy numbers per microfilaria 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 ( P!.001 for both groups), compared with before doxycycline treatment. At this time point, proportions of patients with no microfilaria and no ftsz copies were 5% and 7% in the doxycycline plus placebo diethylcarbamazine-albendazole 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 ( P!.05), compared with before doxycycline treatment (table 2). Body temperature and adverse reactions after diethylcarbamazine-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 temperature 138 C. The effect of doxycycline on adverse reactions to diethylcarbamazine-albendazole was clear from the significantl lower body temperatures and adverse reactions in doxycycline plus diethylcarbamazine-albendazole recipients, compared with those in placebo doxycycline plus diethylcarbamazine-albendazole recipients (figu e 3). The percentage of persons with body temperature 138 C or severe adverse reactions was significantl higher in the placebo doxycycline plus diethylcarbamazine-albendazole group (26.3% and 42.9%, respectively), compared with the doxycycline plus diethylcarbamazine-alben- 1390 CID 2008:46 (1 May) Supali et al.
Figure 3. Body temperature (A) and adverse reactions (B) following diethylcarbamazine-albendazole treatment in groups treated with doxycycline plus placebo diethylcarbamazine-albendazole (DOX & pl DEC), doxycycline plus diethylcarbamazine-albendazole (DOX & DEC), and placebo doxycycline plus diethylcarbamazine-albendazole (pl DOX & DEC). Each mark represents 1 person; horizontal lines represent medians. *Differences between body temperature and adverse reactions in treatment groups were assessed using Kruskal-Wallis nonparametric analysis of variance. dazole group (10.5% and 15.8%, respectively; ble 3). DISCUSSION P!.05) (ta- Doxycycline treatment depleted Wolbachia from B. malayi in infected patients; this is in agreement with studies of bancroftian filariasi [11 13]. Levels of Wolbachia measured before doxycycline treatment, as determined by copy numbers of the ftsz gene per microfilari (median, 300 copies/microfilaria) agree well with copy numbers determined for the wsp gene in a laboratory strain of B. malayi [19]. After doxycycline treatment, the Wolbachia load per microfilari 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 predoxycycline levels. Our results have established that a 6-week course of 100 mg doxycycline per day leads to a strong reduction in microfila - emia in B. malayi infected subjects. Moreover, diethylcarbamazine-albendazole without doxycycline induced a significan reduction in microfila emia; however, only 27% of subjects were amicrofila emic 1 year after treatment, compared with much higher rates of amicrofila emia in the doxycycline plus diethylcarbamazine-albendazole (87.5%) or the doxycycline plus placebo diethylcarbamazine-albendazole (77%) groups. Here, we have also shown that microfilari count as well as prevalence of microfila emia at 1 year after treatment was similar in participants receiving doxycycline plus diethylcarbamazine-albendazole and in those receiving doxycycline plus placebo diethylcarbamazine-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 sufficien to deplete Wolbachia from filaria worms and to lead to almost complete amicrofila emia at 1 year after treatment. Although the measurement of adult worm burden is not possible for brugian filariasi (unlike bancroftian filariasis [20, 21], the fact that doxycycline leads to a strong long-term (12- month) reduction in microfila emia (in contrast to 1 dose of diethylcarbamazine-albendazole) suggests that doxycycline either leads to long-term sterilization of adult female Brugia or has substantial macrofilaricida effects. A strong macrofilaricida 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 significan reduction in microfilari counts at 4 months in the placebo doxycycline group, but the microfila emia prevalence remained at 100%. The levels of microfilaria have been shown to fluctuat seasonally [22]. Pretreatment samples had been collected at the end of the rainy season, whereas the samples before diethylcarbamazine-albendazole treatment were collected in the dry season; thus, seasonality may account for the slight reduction in the microfilaria density at 4 months after treatment in the placebo doxycycline group. The seasonal fluctuatio may also account for the significan 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 microfilaria material and Wolbachia into the bloodstream [3, 17]. Because body temperature is a continuous variable and a major adverse reaction to diethylcarbamazine, we also analyzed it separately. In contrast to the placebo diethylcarbamazine-albendazole group, Doxycycline Treatment in Brugian Filariasis CID 2008:46 (1 May) 1391
Table 3. Adverse reactions and elevations in body temperature in groups treated with doxycycline or placebo plus diethylcarbamazine-albendazole or placebo. Measure Doxycycline plus placebo diethylcarbamazine-albendazole Data are proportion (%) of patients Doxycycline plus diethylcarbamazine-albendazole Placebo doxycycline plus diethylcarbamazine-albendazole 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 score Mild 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 P!.001 for comparison of doxycycline plus placebo diethylcarbamazine-albendazole and doxycycline plus diethylcarbamazine-albendazole groups, by x 2 test. b P!.05 for comparison of doxycycline plus diethylcarbamazine-albendazole and placebo doxycycline plus diethylcarbamazine-albendazole groups, by x 2 test. c P!.01 for comparison of doxycycline plus diethylcarbamazine-albendazole and placebo doxycycline plus diethylcarbamazine-albendazole groups, by x 2 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 diethylcarbamazinealbendazole than in the group who did not receive doxycycline. Moreover, we found that the percentages of persons experiencing 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 antifilaria treatment. In conclusion, finding reported here support the notion that doxycycline, either alone or in combination with antifilaria therapy, strongly reduced Wolbachia levels in B. malayi infected persons and, either alone or in combination with antifilaria therapy, led to a decrease in microfila emia that was sustained for at least 1 year after treatment. Second, doxycycline treatment reduced adverse reactions following diethylcarbamazine-albendazole treatment; this may be particularly important for patients with very high microfilari loads, because they are known to most frequently experience adverse reactions to diethylcarbamazine. The finding are important at 2 levels. At the individual level, doxycycline might be a suitable alternative for individual treatment and for those wishing to avoid adverse reactions to standard treatments. At the community level, although doxycycline is not appropriate for mass drug administration, our finding 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 Gorontalo for their encouragement and help during the trial. We thank Pfize for providing drug treatment free of charge. Financial support. The European Union (European Commission, INCO programme, contract no. IC4-CT-2002-10051). Potential conflict of interest. All authors: no conflicts References 1. Ottesen EA. The global programme to eliminate lymphatic filariasis Trop Med Int Health 2000; 5:591 4. 2. Ottesen EA, Duke BO, Karam M, Bebbehani K. Strategies and tools for the control/elimination of lymphatic filariasis Bull World Health Organ 1997; 75:491 503. 3. Molyneux DH, Bradley M, Hoerauf A, Kyelem D, Taylor MJ. Mass drug treatment for lymphatic filariasi and onchocerciasis. Trends Parasitol 2003; 19:516 22. 4. Noroes J, Dreyer G, Santos A, Mendes VG, Medeiros Z, Addis D. Assessment of the efficac of diethylcarbamazine on adult Wuchereria bancrofti in vivo. Trans R Soc Trop Med Hyg 1997; 91:78 81. 5. Taylor MJ, Bandi C, Hoerauf AM, Lazdins J. Wolbachia bacteria of filaria nematodes: a target for control? Parasitol Today 2000; 16:179 80. 6. Townson S, Hutton D, Siemienska J, et al. Antibiotics and Wolbachia in filaria nematodes: antifilaria activity of rifampicin, oxytetracycline and chloramphenicol against Onchocerca gutturosa, Onchocerca lienalis, and Brugia pahangi. Ann Trop Med Parasitol 2000; 94:801 16. 7. Langworthy NG, Renz A, Mackenstedt U, et al. Macrofilaricida activity of tetracycline against the filaria nematode, Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship. Proc Biol Sci 2000; 267:1063 9. 8. Chirgwin SR, Coleman SU, Porthouse KH, Nowling JM, Punkosdy GA, Klei TR. Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus). Infect Immun 2003; 71:6986 94. 9. Chirgwin SR, Nowling JM, Coleman SU, Klei TR. Brugia pahangi and Wolbachia: the kinetics of bacteria elimination, worm viability, and host responses following tetracycline treatment. Exp Parasitol 2003; 103:16 26. 1392 CID 2008:46 (1 May) Supali et al.
10. Hoerauf A, Mand S, Adjei O, Fleischer B, Büttner DW. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermi after ivermectin treatment. Lancet 2001; 357:1415 6. 11. Hoerauf A, Mand S, Fischer K, et al. Doxycycline as a novel strategy against bancroftian filariasis-depletio of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilari production. Med Microbiol Immunol 2003; 192:211 6. 12. Turner JD, Mand S, Debrah AY, et al. A randomized, double-blind clinical trial of a 3-week course of doxycycline plus albendazole and ivermectin for the treatment of Wuchereria bancrofti infection. Clin Infect Dis 2006; 42:1081 9. 13. Taylor MJ, Makunde WH, McGarry HF, Turner JD, Mand S, Hoerauf A. Macrofilaricida activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomised placebo-controlled trial. Lancet 2005; 365:2116 21. 14. Debrah AY, Mand S, Specht S, et al. Doxycycline reduces plasma VEGF- C/sVEGFR-3 and improves pathology in lymphatic filariasis PLoS Pathog 2006; 2:e92. 15. Michael E, Bundy DA, Grenfell BT. Re-assessing the global prevalence and distribution of lymphatic filariasis Parasitology 1996; 112:409 28. 16. Ottesen EA. Efficac of diethylcarbamazine in eradicating infection with lymphatic-dwelling filaria in humans. Rev Infect Dis 1985;3: 341 56. 17. Cross HF, Haarbrink M, Egerton G, Yazdanbakhsh, Taylor MJ. Severe reactions to filaria chemotherapy and release of Wolbachia endosymbionts into blood. Lancet 2001; 358:1873 5. 18. Haarbrink M, Terhell AJ, Abadi GK, Mitsui Y, Yazdanbakhsh M. Adverse reactions following diethylcarbamazine (DEC) intake in endemic normals, microfilaraemics and elephantiasis patients. Trans R Soc Trop Med Hyg 1999; 93:91 6. 19. McGarry HF, Egerton GL, Taylor MJ. Population dynamics of Wolbachia bacterial endosymbionts in Brugia malayi. Mol Biochem Parasitol 2004; 135:57 67. 20. Taylor MJ, Hoerauf A. A new approach to the treatment of filariasis Curr Opin Infect Dis 2001; 14:727 31. 21. Weil GJ, Lammie PJ, Richards FO Jr, Eberhard ML. Changes in circulating parasite antigen levels after treatment of bancroftian filariasi with diethylcarbamazine and ivermectin. J Infect Dis 1991; 164:814 6. 22. Sartono E, Lopriore C, Kruize YC, Kurniawan-Atmadja A, Maizels RM, Yazdanbakhsh M. Reversal in microfilaria density and T cell responses in human lymphatic filariasis Parasite Immunol 1999; 21:565 71. 23. Rao RU. Endosymbiotic Wolbachia of parasitic filaria nematodes as drug targets. Indian J Med Res 2005; 122:199 204. 24. Stolk WA, de Vlas SJ, Habbema JD. Anti-Wolbachia treatment for lymphatic filariasis Lancet 2005; 365:2067 8. Doxycycline Treatment in Brugian Filariasis CID 2008:46 (1 May) 1393