Doxycycline Treatment of Brugia malayi –Infected Persons Reduces Microfilaremia and Adverse Reactions after Diethylcarbamazine and Albendazole Treatment

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  Doxycycline Treatment of Brugia malayi –Infected Persons Reduces Microfilaremia and Adverse Reactions after Diethylcarbamazine and Albendazole Treatment
  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 Sartono 2 1 Department of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia; Departments of  2 Parasitology and  3 MedicalStatistics 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 efficacy 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 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 all P  ! .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   speciescauselymphaticfilariasis,which affects 120 million people worldwide [1]. Filarialdisease is associated with episodes of acute and chronicinflammation that can lead to conditions such as ele-phantiasis and hydrocele. The global lymphaticfilariasis 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( 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 aretoo low to sustain transmission of filarial parasites inhumans and (2) to reduce morbidity associated withchronic filarial disease [1]. Current strategies involvethe community-based single oral treatment with com-bination of diethylcarbamazine or ivermectin and al-bendazole, which needs to be repeated annuallyorsem-iannually over many years to reduce transmission [2,3]. Additional control strategies are needed, becausethecurrently used drugs show only partial activity againstadult parasites [4].The discovery of   Wolbachia   in filarial specieshaspro-vided a new target for chemotherapy of humanfilariasis  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 rendersadult female worms infertile [8, 9]. In human onchocerciasis,treatment with doxycycline (100 mg/day) for 6 weeks depletedthe 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  DNAlevels. Twelve months after the commencement of doxycyclinetherapy, there was an almost complete absence of microfilar-emia [11, 12]. Recently, it was established that administrationof 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, itis essential to determine whether doxycycline in combinationwith 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 communitiesbecause of adverse reactions of microfilaria-positive subjects tothis drug [16]. There is evidence that adverse reactions to di-ethylcarbamazine coincide with the release of   Wolbachia   intothe bloodstream [17]. It is therefore important to assess theeffect 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 brugianfilariasis as proof of principle that targeting the endosymbiontof   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 Europeanand Indonesian institutions was approved by the Commissionof Medical Ethics of the University of Indonesia (Jakarta, In-donesia) and for European centers by the Research EthicsCom-mittee of the Liverpool School of Tropical Medicine (Liverpool,United Kingdom). The trial registration number is ISRTN37962059. Objectives.  The primary objective was to assess whether a6-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 standardantifilarialdrugs. Study site and enrollment.  The study was performed fromMarch 2002 through August 2003 in Parigi-Moutong, CentralSulawesi, and in Bonebolanggo, Northern Sulawesi, areas of Indonesia where  B. malayi   is endemic. Before the trial, theprevalences of microfilariae in the villages in Parigi-Moutongand Bonebolanggo were 24% and 38%, respectively. The in-habitants were informed of the purpose of the study. Informedconsent was obtained from all persons (or parents) before theclinical 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,  1 5 microfilaria/mL; and good healthwithout any clinical condition requiring long-term use of medications.The study included 161 participantswithmicrofilaremia.Themedian age for men was 29 years (range, 12–76 years) and forwomen was 28.5 years (range, 12–70 years). Exclusion criteriafor all participants were abnormal hepatic and renal profiles(alanine aminotransferase level,  1 30 U/L;  g -glutamyl transpep-tidase level,  1 28 U/L; creatinine level,  1 1.2 mg/dL) measuredby dipstick chemistry (Reflotron; Roche Diagnostics), and ad-ditional exclusion criteria for women included pregnancy orbreast-feeding. Interventions and randomization.  Doxycycline (100 mg)and matched placebo capsules were provided by Pfizer. Dieth- ylcarbamazine, albendazole, and placebo tabletswerepurchasedfrom Indo Farma Pharmaceutical Company.To prevent mix-up of the drugs and matching placeboswithin 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 wereconcealed from investigators and patients. Groups I and IIreceived 1 capsule of doxycyline (100 mg) per day for 6 weeks;group III received 1 capsule of matching placebo per day for6 weeks (figure 1). Drugs were taken under supervision of themedical team, and adverse reactions were monitored and re-corded in a questionnaire completed by medical staff. Duringthese 6-week courses of doxycycline treatment, the patientswere visited by primary health care medical staff who wereunaware of treatment allocation daily.Four months after the start of treatment, groups II and IIIreceived a single dose of diethylcarbamazine (6 mg/kg) plusalbendazole (400 mg), whereas group I received placeboinsteadof diethylcarbamazine and albendazole. For diethylcarbama-zine-albendazole and placebo diethylcarbamazine-albendazoletreatment, all patients were admitted to a primary health centerfor 3 days, which allowed adverse reactions to be monitoredclosely and frequently (twice daily) and for appropriate care tobe provided to the patients. Tablets were swallowed under su-pervision after a meal. Heart rate, blood pressure, and body            F         i       g       u       r       e         1  .       D    e    s      i    g    n    o      f    a    s     t    u      d    y    o      f      d    o    x    y    c    y    c      l      i    n    e     t    r    e    a     t    m    e    n     t    p      l    u    s     t    r    e    a     t    m    e    n     t    w      i     t      h      d      i    e     t      h    y      l    c    a    r      b    a    m    a    z      i    n    e   -    a      l      b    e    n      d    a    z    o      l    e    a    m    o    n    g    p    e    r    s    o    n    s      i    n      f    e    c     t    e      d    w      i     t      h       B    r    u    g      i    a    m    a      l    a    y      i .  1388  •  CID 2008:46 (1 May)  •  Supali et al. Table 1. Microfilaraemia before and after treatment with doxycycline (DOX) and/or diethylcarbamazine-albendazole (DEC). Group, parameterTime relative to treatmentsBefore DOX2 monthsafter DOX4 months after DOX(before DEC) a 3 daysafter DEC7 daysafter DEC1 yearafter DOX a DOX plus placebo DECNo. 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 DECNo. 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 DECNo. 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 for comparison of microfilaria count between treatment groups using Kruskal-Wallis nonparametric analyses of variance. P  ! .001 b , compared with predoxycycline value (Wilcoxon signed rank test). P  ! .001 c , compared with predoxycycline value (Wilcoxon signed rank test). P  ! .05 d , 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 microfilariaden-sity by 50% after 4 months (power, 80%;  a p 0.05), and a15% dropout rate throughout the trial period. For assessmentof the potential reduction in adverse reactions to treatmentwith 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 personsin the control group have elevated proinflammatory cytokinelevels, compared with none of the doxycycline-treated persons(power, 80%;  a p 0.05). Outcomes.  The primary outcome measurements were thequantity of   Wolbachia   single-copy genes per microfilaria (as asurrogate for numbers of whole bacteria per microfilaria) 4months 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 themicrofilaria level were determined by collecting 5 mL of nightblood (obtained between 8:00 and 10:00  p.m. ) in an EDTA-containing tube. One mL of blood was filtered through a 5- m m–pore filter (Millipore) and stained with Giemsa to quantify microfilariae. The filtration and counting of the microfilariaewas performed by the same person on all occasions. Theplasmafrom the rest of the blood was collected by centrifugation andstored at   20  C for serological assays. The remaining bloodpellet was diluted with 5 mL of distilled water and filtered toretain 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 fromblood 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 MgCl 2 , 200  m mol/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 m L of DNA in a 20- m L total reaction volume. Amplificationtook place in a Rotorgene 3000 (Corbett Research) under thefollowing 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 thestartofdoxycyclinetreat-ment or just before diethylcarbamazine-albendazoletreatment;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 pointswereawarded 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 wereawarded5 points, and temperatures  1 38.5  C were awarded 10 points.  Doxycycline Treatment in Brugian Filariasis  •  CID 2008:46 (1 May)  •  1389 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-albendazoletreat-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 baseline P   !  .001predoxycycline levels, determined using the Wilcoxon signed rank test. Adverse reactions were classified into 3 categories exactly asdescribed elsewhere [18]. Statistical analysis.  Statistical analysis was performed inSPSS for Windows, version 11 (SPSS). Differences betweenvar-iables for 3 groups were assessed by use of the Kruskal-Wallistest, whereas the Mann-Whitney  U   test was used for comparingdifferences between 2 groups. A nonparametric, repeated-mea-sure trend test was performed to assess the effect of treatmentover 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 sameindividual were compared using the Wilcoxon matched-pairsrank test. Comparison of proportions was performed using the x 2 test. RESULTS Adverse reactions to doxycycline and diethylcarbamazine- albendazole.  The 6-week course of 100 mg of doxycyline perday was generally well tolerated. The adverse reactionsreportedafter doxycycline administration were mild and tolerable.Noneof the participants had to stop taking doxycycline because of adverse reactions. The adverse effects in the doxycycline group( ) were myalgia ( ), nausea ( ), dizziness n  p 119  n  p 6  n  p 4( ), headache ( ), insomnia ( ), abdominal pain n  p 5  n  p 3  n  p 2( ), itching ( ), diarrhea ( ), rash ( ), mal- n  p 2  n  p 2  n  p 1  n  p 1aise ( ), and drowsiness ( ); in the placebo group n  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 1No report of photosensitivity reactions was recorded during orafter the treatment.The most common adverse reactions to diethylcarbamazine-albendazole were fever, headache, arthralgia, dizziness, lymphnode enlargement, or myalgia. Altogether, 51 of 99 participantsreceiving diethylcarbamazine-albendazole and 8 of 62 receivingplacebo diethylcarbamazine-albendazole experiencedmoderateor severe adverse reactions. Reduction of microfilaria count and prevalence of micro-  filaremia after doxycycline and diethylcarbamazine-alben- dazole treatment.  The microfilaria count before doxycyclinetreatment was the same in all 3 treatment groups. Four monthsafter the start of doxycycline treatment, the microfilaria countin each group was reduced significantly compared with thatbefore doxycycline treatment ( for all groups). The re- P  ! .001duction in microfilaria count was highest in the groups treatedwith doxycycline and lowest in the group treated with placebodoxycycline (89%, 83%, and 39% for the doxycycline plus pla-cebo diethylcarbamazine-albendazole group, the doxycyclineplus diethylcarbamazine-albendazole group, and the placebodoxycycline plus diethylcarbamazine-albendazole group, re-spectively). During diethylcarbamazine-albendazole treatment,there was a strong reduction in microfilaria counts in both
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