• View in gallery
    Figure 1.

    Trial profile of patients examined, randomized, and treated. Of the 57 randomized participants, 43 completed the treatment. One patient was absent for USG examination 7 days after DEC (doxycycline 21 days). At the 12-month follow-up, 6 patients were not present for USG examination (doxycycline 21 days, N = 2; doxycycline 10 days, N = 2; placebo, N = 2) and 15 patients refused to give a blood sample (doxycycline 21 days, N = 8; doxycycline 10 days, N = 3; placebo, N = 4).

  • View in gallery
    Figure 2.

    Representative image of the left supratesticular area. In the two-dimensional mode (inset: lymph vessel with worm nest is circled), the vertical line indicates the slice shown in the one-dimensional m-mode (large image), where filarial movement over time can be seen as undulating lines in the lumen of a dilated lymphatic vessel. Tissue around the vessel where the worm nest is located appears as horizontal lines. The white dashed line indicates the electronic caliper used to measure vessel diameter (marked with a dashed rectangle). In this example, vessel dilation measured from inner layer to inner layer of the lymphatic vessel was 0.61 cm.

  • 1

    WHO, 2007. Global programme to eliminate lymphatic filariasis. Wkly Epidemiol Rec 82 :361–380.

  • 2

    Dreyer G, Addiss D, Williamson J, Noroes J, 2006. Efficacy of co-administered diethylcarbamazine and albendazole against adult Wuchereria bancrofti. Trans R Soc Trop Med Hyg 100 :1118–1125.

    • Search Google Scholar
    • Export Citation
  • 3

    Hussein O, Setouhy ME, Ahmed ES, Kandil AM, Ramzy RM, Helmy H, Weil GJ, 2004. Duplex Doppler sonographic assessment of the effects of diethylcarbamazine and albendazole therapy on adult filarial worms and adjacent host tissues in bancroftian filariasis. Am J Trop Med Hyg 71 :471–477.

    • Search Google Scholar
    • Export Citation
  • 4

    Ottesen EA, Duke BO, Karam M, Behbehani K, 1997. Strategies and tools for the control/elimination of lymphatic filariasis. Bull World Health Organ 75 :491–503.

    • Search Google Scholar
    • Export Citation
  • 5

    WHO, 2004. Towards a strategic plan for research to support the global program to eliminate lymphatic filariasis. Summary of immediate needs and opportunities for research on lymphatic filariasis, Philadelphia, PA, December 9–10, 2003. Am J Trop Med Hyg 71 :1–46.

    • Search Google Scholar
    • Export Citation
  • 6

    Hoerauf A, Mand S, Adjei O, Fleischer B, Büttner DW, 2001. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357 :1415–1416.

    • Search Google Scholar
    • Export Citation
  • 7

    Hoerauf A, Volkmann L, Hamelmann C, Adjei O, Autenrieth IB, Fleischer B, Büttner DW, 2000. Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet 355 :1242–1243.

    • Search Google Scholar
    • Export Citation
  • 8

    Debrah A, Mand S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Büttner M, Adjei O, Büttner DW, Hoerauf A, 2007. Macrofilaricidal activity after four weeks doxycycline treatment of Wuchereria bancrofti. Trop Med Int Health 12 :1433–1441.

    • Search Google Scholar
    • Export Citation
  • 9

    Debrah AY, Mand S, Specht S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Larbi J, Lawson B, Taylor M, Adjei O, Hoerauf A, 2006. Doxycycline reduces plasma VEGF-C/sVEGFR-3 and improves pathology in lymphatic filariasis. PLoS Pathog 2 :e92.

    • Search Google Scholar
    • Export Citation
  • 10

    Turner JD, Mand S, Debrah AY, Muehlfeld J, Pfarr K, McGarry HF, Adjei O, Taylor MJ, Hoerauf A, 2006. 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 42 :1081–1089.

    • Search Google Scholar
    • Export Citation
  • 11

    Hoerauf A, Mand S, Fischer K, Kruppa T, Marfo-Debrekyei Y, Debrah AY, Pfarr KM, Adjei O, Büttner DW, 2003. Doxycycline as a novel strategy against bancroftian filariasis-depletion of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilaria production. Med Microbiol Immunol (Berl) 192 :261–273.

    • Search Google Scholar
    • Export Citation
  • 12

    Dreyer G, Amaral F, Noroes J, Medeiros Z, Addiss D, 1995. A new tool to assess the adulticidal efficacy in vivo of antifilarial drugs for bancroftian filariasis. Trans R Soc Trop Med Hyg 89 :225–226.

    • Search Google Scholar
    • Export Citation
  • 13

    Faris R, Hussain O, El Setouhy M, Ramzy RM, Weil GJ, 1998. Bancroftian filariasis in Egypt: visualization of adult worms and subclinical lymphatic pathology by scrotal ultrasound. Am J Trop Med Hyg 59 :864–867.

    • Search Google Scholar
    • Export Citation
  • 14

    Mand S, Marfo-Debrekyei Y, Dittrich M, Fischer K, Adjei O, Hoerauf A, 2003. Animated documentation of the filaria dance sign (FDS) in bancroftian filariasis. Filaria J 2 :3.

    • Search Google Scholar
    • Export Citation
  • 15

    Debrah A, Mand S, Toliat MR, Marfo-Debrekyei Y, Batsa L, Nurnberg P, Lawson B, Adjei O, Hoerauf A, Pfarr K, 2007. Plasma vascular endothelial growth factor-A (VEGF-A) and VEGF-A gene polymorphism are associated with hydrocele development in lymphatic filariasis. Am J Trop Med Hyg 77 :601–608.

    • Search Google Scholar
    • Export Citation
  • 16

    Hoerauf A, 2008. Filariasis: new drugs and new opportunities for lymphatic filariasis and onchocerciasis. Curr Opin Infect Dis 21 :673–681.

    • Search Google Scholar
    • Export Citation
  • 17

    Debrah AY, Mand S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Lawson B, Taylor M, Adjei O, Hoerauf A, 2009. Reduction in levels of plasma vascular endothelial growth factor-A and improvement in hydrocele patients by targeting endosymbiotic Wolbachia sp. in Wuchereria bancrofti with doxycycline. Am J Trop Med Hyg 80 :956–963.

    • Search Google Scholar
    • Export Citation
  • 18

    Taylor MJ, Makunde WH, McGarry HF, Turner JD, Mand S, Hoerauf A, 2005. Macrofilaricidal activity following doxycycline treatment of Wuchereria bancrofti: a double-blind randomised controlled trial. Lancet 365 :2116–2121.

    • Search Google Scholar
    • Export Citation
  • 19

    Kazura J, Greenberg J, Perry R, Weil G, Day K, Alpers M, 1993. Comparison of single-dose diethylcarbamazine and ivermectin for treatment of bancroftian filariasis in Papua New Guinea. Am J Trop Med Hyg 49 :804–811.

    • Search Google Scholar
    • Export Citation
  • 20

    El Setouhy M, Ramzy RM, Ahmed ES, Kandil AM, Hussain O, Farid HA, Helmy H, Weil GJ, 2004. A randomized clinical trial comparing single- and multi-dose combination therapy with diethylcarbamazine and albendazole for treatment of bancroftian filariasis. Am J Trop Med Hyg 70 :191–196.

    • Search Google Scholar
    • Export Citation
  • 21

    Kshirsagar NA, Gogtay NJ, Garg BS, Deshmukh PR, Rajgor DD, Kadam VS, Kirodian BG, Ingole NS, Mehendale AM, Fleckenstein L, Karbwang J, Lazdins-Helds JK, 2004. Safety, tolerability, efficacy and plasma concentrations of diethylcarbamazine and albendazole co-administration in a field study in an area endemic for lymphatic filariasis in India. Trans R Soc Trop Med Hyg 98 :205–217.

    • Search Google Scholar
    • Export Citation
  • 22

    Sunish IP, Rajendran R, Mani TR, Dash AP, Tyagi BK, 2006. Evidence for the use of albendazole for the elimination of lymphatic filariasis. Lancet Infect Dis 6 :125–126.

    • Search Google Scholar
    • Export Citation
  • 23

    Hoerauf A, Specht S, Buttner M, Pfarr K, Mand S, Fimmers R, Marfo-Debrekyei Y, Konadu P, Debrah AY, Bandi C, Brattig N, Albers A, Larbi J, Batsa L, Adjei O, Buttner DW, 2008. Wolbachia endobacteria depletion by doxycycline as antifilarial therapy has macrofilaricidal activity in onchocerciasis: a randomized placebo-controlled study. Med Microbiol Immunol (Berl) 197 :295–311.

    • Search Google Scholar
    • Export Citation
  • 24

    Santos MD, Vermeersch H, Remon JP, Schelkens M, De Backer P, Van Bree HJ, Ducatelle R, Haesebrouck F, 1996. Pharmacokinetics and bioavailability of doxycycline in turkeys. J Vet Pharmacol Ther 19 :274–280.

    • Search Google Scholar
    • Export Citation
  • 25

    Noroes J, Dreyer G, Santos A, Mendes VG, Medeiros Z, Addiss D, 1997. Assessment of the efficacy of diethylcarbamazine on adult Wuchereria bancrofti in vivo. Trans R Soc Trop Med Hyg 91 :78–81.

    • Search Google Scholar
    • Export Citation
  • 26

    Weil GJ, Ramzy RM, El Setouhy M, Kandil AM, Ahmed ES, Faris R, 1999. A longitudinal study of bancroftian filariasis in the Nile Delta of Egypt: baseline data and one-year follow-up. Am J Trop Med Hyg 61 :53–58.

    • Search Google Scholar
    • Export Citation
  • 27

    Mand S, Debrah A, Batsa L, Adjei O, Hoerauf A, 2004. Reliable and frequent detection of adult Wuchereria bancrofti in Ghanaian women by ultrasonography. Trop Med Int Health 9 :1111–1114.

    • Search Google Scholar
    • Export Citation
  • 28

    Helmy H, Weil GJ, Ellethy AS, Ahmed ES, Setouhy ME, Ramzy RM, 2006. Bancroftian filariasis: effect of repeated treatment with diethylcarbamazine and albendazole on microfilaraemia, antigenaemia and antifilarial antibodies. Trans R Soc Trop Med Hyg 100 :656–662.

    • Search Google Scholar
    • Export Citation
  • 29

    Meyrowitsch DW, Simonsen PE, Magesa SM, 2004. Long-term effect of three different strategies for mass diethylcarbamazine administration in bancroftian filariasis: follow-up at 10 years after treatment. Trans R Soc Trop Med Hyg 98 :627–634.

    • Search Google Scholar
    • Export Citation
  • 30

    Njenga SM, Wamae CN, Njomo DW, Mwandawiro CS, Molyneux DH, 2008. Impact of two rounds of mass treatment with diethylcarbamazine plus albendazole on Wuchereria bancrofti infection and the sensitivity of immunochromatographic test in Malindi, Kenya. Trans R Soc Trop Med Hyg 102 :1017–1024.

    • Search Google Scholar
    • Export Citation
  • 31

    Peixoto CA, Figueiredo-Silva J, 2001. Fine structure of intrascrotal lymphatic vessels infected by Wuchereria bancrofti adult worms. J Submicrosc Cytol Pathol 33 :125–131.

    • Search Google Scholar
    • Export Citation
  • 32

    Dreyer G, Noroes J, Figueredo-Silva J, Piessens WF, 2000. Pathogenesis of lymphatic disease in bancroftian filariasis: a clinical perspective. Parasitol Today 16 :544–548.

    • Search Google Scholar
    • Export Citation
  • 33

    Freedman DO, Bui T, De Almeida Filho PJ, Braga C, Maia e Silva MC, Maciel A, Furtado AF, 1995. Lymphoscintigraphic assessment of the effect of diethylcarbamazine treatment on lymphatic damage in human bancroftian filariasis. Am J Trop Med Hyg 52 :258–261.

    • Search Google Scholar
    • Export Citation
Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 481 242 74
PDF Downloads 120 55 4
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Macrofilaricidal Activity and Amelioration of Lymphatic Pathology in Bancroftian Filariasis after 3 Weeks of Doxycycline Followed by Single-Dose Diethylcarbamazine

Sabine MandInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Sabine Mand in
Current site
Google Scholar
PubMed
Close
,
Kenneth PfarrInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Kenneth Pfarr in
Current site
Google Scholar
PubMed
Close
,
Prakash K. SahooInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Prakash K. Sahoo in
Current site
Google Scholar
PubMed
Close
,
Ashok K. SatapathyInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Ashok K. Satapathy in
Current site
Google Scholar
PubMed
Close
,
Sabine SpechtInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Sabine Specht in
Current site
Google Scholar
PubMed
Close
,
Ute KlarmannInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Ute Klarmann in
Current site
Google Scholar
PubMed
Close
,
Alexander Y. DebrahInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Alexander Y. Debrah in
Current site
Google Scholar
PubMed
Close
,
Balachandran RavindranInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Balachandran Ravindran in
Current site
Google Scholar
PubMed
Close
, and
Achim HoeraufInstitute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Bonn, Germany; Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India; Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, India; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana

Search for other papers by Achim Hoerauf in
Current site
Google Scholar
PubMed
Close

In a placebo controlled trial, the effects of 21- and 10-day doxycycline treatments (200 mg/day) followed by single dose diethylcarbamazine (administered 4 months post treatment) on depletion of Wolbachia endobacteria from Wuchereria bancrofti, filaricidal activity, and amerlioration of scrotal lymph vessel dilation were studied in 57 men from Orissa, India. The 21-day doxycycline course reduced Wolbachia in W. bancrofti by 94% before diethylcarbamazine administration. After 12 months, all patients with this treatment were amicrofilaremic and different from the 10-day doxycycline (42.9%) and placebo (37.5%) groups, and significantly fewer were positive for scrotal worm nests (6.7%) compared with 10-day doxycycline (60%) and placebo (66.7%). Average scrotal lymph vessel diameters were reduced from 0.7 cm pre-treatment to 0.02 cm in patients after 21 days of treatment, while no significant changes were seen in the other groups. This latter feature confirms the beneficial effects of doxycycline on lymphatic dilation and thus adds to the existing evidence that doxycycline, in addition to being macrofilaricidal, may be used to prevent or reverse lymphatic pathology.

INTRODUCTION

The community-based control of lymphatic filariasis (LF) is aimed at interrupting parasite transmission.1 The antifilarial drugs currently used, diethylcarbamazine (DEC), albendazole, and ivermectin, are predominantly active against microfilariae (Mf), with DEC showing partial activity against adult worms.24 Because the adult filariae cause pathologies such as dilation of scrotal lymphatics, hydrocele, and lymphedema, and because current antifilarial drugs do not cure adult worm infection, research is needed to provide novel drugs with macrofilaricidal and pathology-improving activity.5 A new approach is the use of antibiotics to deplete Wolbachia endosymbionts that was shown to lead to inhibition of worm embryogenesis and eventually viability in human onchocerciasis. 6,7 In LF, 6-week and 4-week doxycycline treatments (200 mg/day) are effective at reducing Wolbachia single gene copies/Mf by 95%, which leads to sustained reductions of microfilaremia and shows strong macrofilaricidal activity of > 90% with concurrent improvement of LF pathology. 8,9 However, the duration of this treatment is rather long, even for the treatment of individuals or in selected problem areas, making a shorter regimen with doxycycline, likely in combination with “standard” antifilarial drugs, advantageous.

A 3-week course of doxycycline treatment followed by a single dose of ivermectin plus albendazole showed a reduction in Wolbachia single gene copies/Mf of ~80% and led to a sustained absence of microfilaremia for > 1 year 10; however, no macrofilaricidal activity was observed with this regimen. Because most LF pathologies are induced by adult worms rather than Mf, it is important to determine the threshold, or minimum treatment duration of doxycycline in combination with one of the classically used drugs that retains macrofilaricidal activity and improves lymphatic pathology. In search of such a regimen, we conducted a placebo-controlled study in W. bancrofti–infected individuals in Orissa, India to verify or reject a macrofilaricidal effect of 3 weeks of doxycycline in combination with DEC as an antifilarial treatment.

MATERIALS AND METHODS

Study population.

A placebo-controlled, double-blind study was carried out in communities in the state of Orissa, India. The study was approved by the Ethical Committee of the Regional Medical Research Center, Bhubaneswar, India, as well as the Internal Review Board of the University Hospital Bonn, and conformed to the principles of the Helsinki Declaration of 1964 (latest amendment 2004). Individuals were residents of the villages of Bhimpur, Upper Basta, Kansapada, Podapada, and Padanpur in the district Orissa. The study site was selected based on the prevalence of LF, which had been assessed before by clinical observations (rapid assessment of patients with hydrocele and lymphedema to confirm endemicity of LF). A total of 57 male asymptomatic Mf carriers (Figure 1) were included in this study. Oral informed consent was obtained from all participants after detailed explanation of the study in the local language and in the presence of two witnesses. Participants were eligible for the study if they were men 18–50 years of age, had a minimum body weight of 40 kg, were in good health, and were without any clinical condition requiring long-term medication. Hepatic and renal functions were assessed by dipstick chemistry. Exclusion criteria encompassed abnormal hepatic and renal enzymes (aspartate aminotransferase > 0–40 IU/L, alanine aminotransferase > 0–45 IU/L, and creatinine 53–126 μmol/L), intolerance to doxycycline or DEC, and history of alcohol or drug abuse.

Randomization, treatment regimens, and follow-up.

Participants were randomly allocated to the three treatment groups using computer-assisted randomization and received 200 mg doxycycline (2 capsules, 100 mg each; Dr. Reddy’s Laboratories, Hyderabad, India) either for 21 or 10 days, followed by 11 days matching placebo or they only received matching placebo for 21 days. The treatment was monitored daily and directly by a trial clinician. All participants received a single dose of DEC (6 mg/kg; GlaxoSmithKline Pharmaceuticals, Mumbai, India) 4 months after the doxycycline and placebo treatment courses had been initiated.

Patients were followed-up with ultrasonography at the points of time shown in Table 2. Blood was taken for Mf determination at the points of time shown in Table 1, and Wolbachia gene copy numbers were quantified using polymerase chain reaction (PCR) before treatment and after 4 months but before the administration of DEC.

Determination of microfilaremia.

Before the initiation of doxycycline treatment, the Mf load was assessed by microscopic examination of finger prick night blood samples as published. 10 Subsequently, eligible patients donated 10 mL of venous blood for accurate Mf quantification using the Whatman Nucleopore filter method as described previously. 11 Afterward, using a microscope and fine glass pipette, 100 Mf were picked from the remaining blood and lyophilized for later DNA extraction. At follow-up, 7–10 mL of blood were taken from patients. At each time point, plasma was frozen for later analysis of antigenemia.

Determination of circulating filarial antigen.

Circulating filarial antigen (CFA) was measured with the TropBio ELISA test kit (TropBio, Townsville, Australia). The manufacturer’s protocol was followed except that samples were diluted 1:20 in sample diluent before boiling. Fifty microliters of diluted liquid was added to plate wells, and wells were incubated overnight. Optical density at 414 nm was recorded from plasma samples taken at the time points listed above. Antigen units were calculated with a standard curve from standards provided by the manufacturer.9

Assessment of Wolbachia levels in microfilariae by PCR.

Wolbachia levels per microfilaria were quantified by real-time PCR of the W. bancrofti WolbachiaftsZ gene (AF081198), derived from 100 Mf before and 4 months after treatment. DNA was extracted with the QIAamp kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol with Proteinase K digestion. For quantification, primers and a Taqman hybridization probe with the fluorescent dye 6-FAM (6-carboxyfluorescein; Qiagen) were used to amplify a 286-bp fragment of the W. bancrofti Wolbachia-ftsZ (accession number AF081198). Primers used were as follows—FtsZ_F1: 5′-GCTTGGGCTTGAAGAATTACAAA-3′; FtsZ_R1: 5′-GCTATCAGTGCTGCAGAAGC-3′;and FtsZ_hybridization: 5′-ACGTAGACACGCTTATTGTCATTCCAAACCA-3′. Cycling conditions were 15 minutes at 95°C, followed by 40 cycles of 60°C with signal acquired on the FAM channel. The products were quantified by comparing with a standard curve of the plasmid containing the W. bancrofti Wolbachia-ftsZ fragment, using a Rotor-Gene 3000 (Corbett Research, Brussels, Belgium) real-time cycler. 11

Assessment of worm nests and dilation of lymphatic vessels at worm nest location and in the supratesticular area.

To assess the macrofilaricidal activity of the treatment, the number of worm nests in the scrotal lymphatic vessels was monitored using ultrasonography (USG). USG examinations were performed before and 4 months after the onset of doxycycline/placebo treatment (before DEC administration), 24 hours and 7 days after a single dose of DEC, and 12 months after doxycycline/placebo treatment initiation. The ultrasonographer was blinded to the nature of the treatment. Examinations were done using the portable ultrasound system SonoSite 180 Plus (SonoSite, Erlangen, Germany), equipped with an L 38-mm 5- to 10-MHz linear array transducer. Worm nests were detected by the typical movement pattern of filarial worms, the so-called filaria dance sign (FDS), in the two-dimensional b-mode 12 and confirmed using the Pulse Wave Doppler mode. 13,14 Dilation of the scrotal lymphatic vessels at worm nest location was measured in the one-dimensional m-mode from inner layer to inner layer of the lymphatic vessel (Figure 2). 14 To determine the stage of dilation in the supratesticular lymphatic vessels, the maximum dilation was measured in b-mode. Documentation of findings was performed using a Handycam (Sony, Tokyo, Japan).

Statistical analyses.

For microfilaremia, antigenemia, and Wolbachia levels (Wolbachia ftsZ copy numbers/Mf), geometric mean and median with 10th and 90th percentiles were calculated. For scrotal lymphatic vessel dilation and worm nest number, the mean ± SD and median with range were calculated. Differences at baseline and subsequent follow-ups were analyzed using the following tests: analysis of variance (ANOVA) post hoc test, Student t test, unpaired t test, Wilcoxon signed rank test, Friedman test, and Fisher exact test. P < 0.05 was considered significant. All analyses were done with the program Stat View for Mac, software version 5.0.

RESULTS

Of 126 volunteers examined, 57 asymptomatic male microfilaria carriers were included in the trial and allocated to one of the three different treatment arms. The three regimens administered were 1) treatment of 21 days with 200 mg doxycycline/day; 2) treatment of 10 days with 200 mg doxycycline/day followed by 11 days matching placebo; and 3) administration of placebo for 21 days. Forty-three patients completed the whole treatment course (Figure 1).

Microfilaremia.

All patients were microfilaremic before treatment start (Table 1). Four months after treatment onset and before DEC administration, 84% of all participants were still Mf positive, although the number of Mfs was slightly reduced in all groups. The subsequent single dose of DEC led to a significant drop of Mf numbers in all groups 24 hours after intake, which persisted in all groups and was more pronounced at the 12-month follow-up. Interestingly, the combination of 21-day doxycycline and DEC apparently led to a strong, if not complete, inhibition of embryogenesis, because all patients were amicrofilaremic at 12 months, whereas in 42.9% of the 10-day doxycycline group and 37.5% of the placebo group, Mfs could be detected at 12 months (Table 1).

Wolbachia levels in microfilariae.

Wolbachia levels in Mfs were measured before treatment onset and 4 months thereafter, but before DEC administration. There was no difference in the Wolbachia levels in Mfs between the three groups before treatment (ANOVA post hoc test, unpaired t test). Four months after doxycycline/placebo treatment, before DEC administration, there was a significant reduction by 94% in the 21-day doxycycline group. Interestingly, Wolbachia loads were also reduced by 90.4% in the 10-day doxycycline group; however, the reduction was not as homogenous as in the 21-day group and therefore did not reach statistical significance (Table 1). In the placebo group, a trend to an increase of Wolbachia per Mf could be observed (+64.1%). Accordingly, the unpaired t test showed a significant difference for the 21-day doxycycline group versus placebo (P = 0.01) and a strong trend between the 10-day doxycycline group versus placebo (P = 0.06), whereas no significant difference could be seen between the two doxycycline groups (P = 0.6).

Antigenemia.

Before treatment, there was no significant difference in antigen levels between the three groups (unpaired t test). In parallel to the reduction of microfilaremia, CFA levels were reduced at the 12-month time point in all groups. However, there was a strong tendency to lower CFA levels (P = 0.0503, Friedman test) in the 21-day doxycycline group compared with the reductions seen in the 10-day doxycycline and the placebo groups (P = 0.4119 and 0.1357, respectively).

FDS and number of worm nests per patient.

The number of worm nests in patients was comparable in all groups before treatment (means, 2.12, 2.08, and 2.17 for 21 days, 10 days, and placebo, respectively; unpaired t test), and there were no significant changes at 4 months. Seven days after DEC, in the 21-day doxycycline group, there was a significant drop in the proportion of FDS-positive patients and in worm nests detected compared with 24 hours after DEC (P = 0.04, Wilcoxon signed rank test). This was not observed in the 10-day and placebo groups when these two time points were compared (P = 0.42 and P = 0.5, respectively; the significance in Table 2 depicts the comparison to pre-treatment). This suggests that the 21-day doxycycline regimen had already delivered some sort of damage to the worms that led to their death after DEC administration.

The long-term follow-up at 12 months showed that in 14 of 15 patients (93.3%) in the 21-day doxycycline group, no FDS could be found, concomitant with a > 20-fold reduction in the number of worm nests (P = 0.0001, Friedman test). A definite loss of worm nests was also observed in the 10-day doxycycline group, with 40% of the patients negative for FDS. However, it was clearly less pronounced compared with the 21-day doxycycline group and significant only when all time points were included in the Friedman test; 33.3% of the participants in the placebo group were negative for FDS, but this was not significant. When comparing between treatment groups, there were significant differences in worm nest loss between the 21-day doxycycline and the 10-day doxycycline groups (P = 0.005, unpaired t test); between the 21-day doxycycline and the placebo groups (P = 0.006, unpaired t test); and between patients being positive or negative for FDS (P = 0.007 and P = 0.003, respectively; Fisher exact test). In contrast, there were no significant differences between the 10-day doxycycline and the placebo group (P = 0.96, unpaired t test; P > 0.99, Fisher exact test). Thus, significant macrofilaricidal effects greater than those seen with DEC alone were only achieved in the 21-day doxycycline group.

Dilation of supratesticular lymphatic vessels at worm nest location.

Dilation of the scrotal lymphatic vessels was evenly distributed in all three groups before onset of treatment (means, 0.71, 0.67, and 0.79 cm, respectively; unpaired t test). Similar to the reduction in the number of worm nests in the 21-day doxycycline group at 4 months and, most notably, at 12 months, there was also a significant decrease in the dilation of the scrotal lymphatic vessels. In the 10-day doxycycline group, there was also a significant drop when assessment was done using the Friedman test, probably arising from the transient increase in vessel dilation at the 4-month time point. When only an endpoint analysis of the long-term reduction in vessel dilation was considered and compared with pre-treatment, differences between the 10-day doxycycline and the placebo groups were no longer significant. Comparing the three treatment groups, there was a significant difference between the 21-day doxycycline and 10-day doxycycline groups (P = 0.024) and between the 21-day doxycycline and placebo groups (P = 0.02), but no difference between the 10-day doxycycline and placebo groups (unpaired t test).

Assessment of adverse reactions.

No serious adverse events occurred during the period of doxycycline/placebo treatment or DEC administration. Side effects reported, such as diarrhea, headache, or joint pain, were always mild and did not occur more frequently in the verum arms compared with placebo. All adverse events were treated symptomatically by the trial clinician, using, where necessary, oral rehydration salt, paracetamol, or diclofenac tablets. None of the complaints lasted for > 3 days.

Adverse reactions after DEC administration are summarized in Table 3. As with doxycycline, patients received oral rehydration salt, paracetamol, or diclofenac tablets from the trial clinician where necessary and were monitored until resolution of the complaints. There was no significant difference between the three treatment groups in the number and severity of side effects 24 hours and 7 days after DEC administration when these events were simply enumerated and compared between the treatment groups using the unpaired t test.

DISCUSSION

Doxycycline is a safe and efficacious drug for treatment of W. bancrofti. Although it requires a longer treatment time than currently used drugs, the advantage of anti-wolbachial treatment is that it has shown higher macrofilaricidal activity than DEC (ivermectin is not macrofilaricidal on its own) and that it leads to a reduction of lymph vessel dilation as well as to an improvement of lymphedema stages 9,15,16 and also hydrocele. 17 Because the treatment time (4 weeks) with just doxycycline needed to achieve a macrofilaricidal effect is long, the macrofilaricidal efficacy of doxycycline treatment before DEC treatment was tested. Two time courses (10 and 21 days) of doxycycline were included in the study design to determine whether the combination treatment would allow for a shorter doxycycline treatment.

A 21-day doxycycline 200-mg/day regimen was compared with 10-day doxycycline 200 mg/day and a placebo group; all groups received DEC 6 mg/kg 4 months after the onset of doxycycline treatment. Both drugs, doxycycline and DEC, are known to have macrofilaricidal activity to different extents in bancroftian filariasis. Besides an adulticidal effect, doxycycline has recently been shown to ameliorate pathology in patients with bancroftian filariasis, such as dilation in supratesticular scrotal lymphatic vessels, lymphedema, and hydrocele.9 This effect was previously unknown and has not been observed after DEC; rather, in contrast, transient hydroceles developed and pre-existing ones increased after DEC.3 We therefore addressed the following parameters in this trial: 1) long-term antimicrofilarial activity (caused by sterilization of adult female worms); 2) depletion of Wolbachia; 3) loss of worm nests; 4) reduction of antigen levels; and 5) amelioration of dilated scrotal lymphatic vessels.

Four-month follow-up.

After doxycycline/placebo treatment, all patients received DEC 4 months after the start of the study. Before DEC administration, there was no difference in the number of Mfs between groups. Despite this, a clear and significant 94% reduction in the number of Wolbachia was seen in the 21-day doxycycline group. In the 10-day doxycycline group, a 90% reduction was seen, but because the range was large and not homogenous, this reduction was not significant. No significant differences in antigenemia were measured at this time point, a result expected from the presence of Mfs. However, in the 21-day doxycycline group, macrofilaricidal effects were evident, with a significant drop in worm nests detected and proportion of FDS-positive patients compared with placebo. The reduction in Wolbachia did not lead to a significant reduction in the loss of worm nests, FDS, and in the supratesticular lymphatic vessel dilation before DEC administration. From previously published studies, the results for the 4-month follow-up are not surprising, because we showed that Mf levels decrease according to Mf half-life without DEC or ivermectin intervention, 11 and significant and apparent reductions in worm nests and vessel dilation are more easily seen 12 months after the study start, when the adult worms have died and their antigens, such as CFA, have been cleared. 8,9,17 Because the 10-day doxycycline group did have a 90% reduction in Wolbachia that resulted in loss of worm nests and FDS 7 days after DEC administration, treatment times between 10 and 21 days should be tested to determine the shortest regimen that will result in the macrofilaricidal effects and reverse early pathology.

Twelve-month follow-up.

Long-term antimicrofilarial activity.

Previous trials16 with doxycycline have shown a sustained sterilizing effect on female worms. In Tanzania, an 8-week regimen of 200 mg doxycycline/day resulted in 75% of the patients being Mf negative 15 months after treatment. 18 A 6-week doxycycline 200 mg/day regimen plus a single dose of ivermectin and albendazole in Ghana showed that 94% of the patients were amicrofilaremic in contrast to 50% of the patients who received ivermectin and albendazole alone.9 A further trial in Ghana using 200 mg doxycycline/day for 4 weeks plus a single dose of ivermectin also resulted in only 6% of the doxycycline-treated patients being microfilaremic, whereas all patients of a small control group (7/7) remained microfilaremic at the 12-month follow-up time point.8

Compared with this sustained amicrofilaremia after doxycycline, DEC as single-dose treatment, but also when given as a 7-day course, is inferior and similar to what we observed in the placebo group in this study. Thus, although Kazura and others 19 reported a reduction in microfilaremia of 86–90% 18 months after DEC administration in a population in Papua New Guinea, all patients remained apparently Mf positive. El Setouhy and others 20 compared a 7-day course of DEC (6 mg/kg/day) plus albendazole (400 mg/day) with a single dose of the same combination. Although average Mf levels dropped 99.6% versus 85.7% within a year, complete clearance was only seen in 75% and 23% of the individuals, respectively. Kshirsagar and others 21 showed in India that 67% of the DEC-treated patients were Mf negative 12 months after a single dose. Compared with these results, the 21-day doxycycline course (followed by single-dose DEC) in this study is superior, with none of the individuals staying microfilaremic. In contrast, the 10-day course of doxycycline did not show superiority to single-dose DEC, either when comparing it to the placebo arm in this study or when the comparison is done with the results cited from other studies above.

Depletion of Wolbachia assessed by PCR.

The concept of anti-wolbachial chemotherapy to treat filariasis is based on earlier results of animal models, 22 as well as human clinical trials in onchocerciasis and LF. Depletion of Wolbachia endobacteria in adult female worms results in female worm sterility, 11,23 followed by worm death at longer observation periods.9 Wolbachia depletion was assessed in this study and showed a reduction of 94% (P = 0.002) in the 21-day doxycycline group 4 months after doxycycline/placebo treatment. Interestingly Wolbachia loads were also reduced by 90.4% (P = 0.091) in the doxycycline (10 day) group. The lack of statistical significance indicates a smaller, and more importantly, less homogenous reduction in Wolbachia gene copy number, supporting earlier observations 10 that thresholds exist below which the Wolbachia reduction must fall to achieve the anti-parasitic effects as seen in the 21-day course or as observed in other studies. 8,9 In the placebo group, no significant change of Wolbachia per Mf could be observed. Interestingly, in our earlier study in Ghana 10 where doxycycline was also administered for 3 weeks, Wolbachia copy numbers were only reduced by 80% after 4 months (i.e., before any other combination drug was administered). The reasons for this lower performance of doxycycline in Africa is not clear, but reasons like different nourishment are likely because it is known from systematic studies in animals that these factors result in better pharmacokinetics and bioavailability of doxycycline. 24 Also, the average body weight seemed to be higher in study participants in Africa than in this study in India, although in this trial the body weight was unfortunately not documented.

Macrofilaricidal activity assessed by USG.

Studies using doxycycline with anti-wolbachial activity: A study in Tanzania, where doxycycline at a daily dose of 200 mg/day was administered for 8 weeks without other antifilarial drugs, showed a 78% macrofilaricidal effect 15 months after treatment. 18 In Ghana, a 6-week doxycycline treatment followed by a single dose of ivermectin (150 μg/kg) and albendazole (400 mg) 4 months later showed a 77% macrofilaricidal effect at the 12-month follow-up and 92% after 2 years.9 The 4-week doxycycline (200 mg/day) regimen in Ghanaian participants resulted in a macrofilaricidal effect of 71% after 12 months and in 83% after 24 months.8

Compared with these studies, the 21-day course of doxycycline reported here resulted in an equivalent macrofilaricidal activity (93.3%). In the study in Ghana by Turner and others, 10 doxycycline given for 21 days did not reduce Wolbachia as much as it did in this study, and no macrofilaricidal effect was observed. Whether the macrofilaricidal effect in the 21-day doxycycline group seen in this trial resulted from the combination with DEC, which also has macrofilaricidal activity whereas ivermectin does not, or from the stronger Wolbachia reduction seen before the administration of DEC cannot be decided at present.

Studies using DEC with anti-filarial activity: Earlier trials using DEC and the combination of DEC + albendazole showed diverse results regarding adulticidal effects. A research group from Brazil reported the disappearance of 51.3% of worm nests after a single dose of 6 mg/kg DEC. 25 Similar to this trial, a research group from India reported 53% macrofilaricidal activity in subjects treated with a single dose of DEC 6 mg/kg and placebo (matching albendazole). The combination of DEC 6 mg/kg plus albendazole 400 mg resulted in the loss of 67% of FDS. 21 In contrast to these results, a more recent study showed 77.8% macrofilaricidal activity after DEC 6 mg/kg alone compared with 43.7% after DEC plus albendazole, arguing against an additive effect of the combination of DEC + albendazole.2

Contrary to the above-described studies with DEC alone, we saw only a trend to macrofilaricidal effects in our placebo group (33% worm nest reduction, not significant). This may be because of different follow-up times after DEC treatment. In earlier studies, most prominent adulticidal effects were observed between 1 and 6 months after DEC. In our study, a follow-up was performed 8 months after DEC. It may thus be possible that moderate adulticidal effects of DEC were masked by the appearance of newly acquired worms, because the study area is highly endemic with a high level of ongoing transmission.

Attention should also be paid to a trial conducted in Egypt where single-dose DEC 6 mg/kg resulted in the loss of a total of 87.1% of worm nests in this group, whereas DEC 6 mg/kg for 7 days reduced the total number of active worm nests by 93.8%. With regard to this study, one must take into consideration that the trial took place in a low-endemicity area in Egypt without much transmission where the baseline incidence of Mf was < 1%. 26 A low transmission rate would result in a higher proportion of older worms that are more susceptible to drug action and may explain the better performance of DEC in this study compared with the others, which were carried out in areas of high endemicity (i.e., ongoing high levels of transmission). In addition, volunteers in the Egyptian study also participated in the annual mass treatment on top of receiving the study medication and therefore received the antifilarial drug DEC two times within the 12-month observation period. Finally, these results originated from subjects of both sexes. Because it is known that the detection rate of adult filariae in lymphatic vessels in women is lower than in men, 20,27 the data from this study are not directly comparable to those from the other trials.

Macrofilaricidal activity assessed by circulating filarial antigen.

Remarkable results were obtained regarding the circulating filarial antigen levels determined by the TropBio enzyme assay. In contrast to the 3-week doxycycline trial in Ghana (combination with ivermectin and albendazole) where no difference in CFA levels were observed, 10 in this trial, antigen levels were 65% lower 12 months after treatment (P = 0.0503) in the 21-day doxycycline group. The levels did not decrease significantly in the 10-day doxycycline and the placebo groups. Compared with other clinical trials using doxycycline, 8,9 the decrease at the 12-month follow-up is much stronger in this study, suggesting that the combination of doxycycline with DEC may enhance the macrofilaricidal effects of each individual drug. In the 6-week trial in Ghana, there was no significant decrease of CFA after 12 months, but 24 months after treatment, a significant reduction of 94% was observed.9 In the 4-week trial in Ghana, a decrease of only 5% was observed after 12 months, whereas a reduction of 88% could be seen after 24 months.8 It therefore seems that the 12-month time point may have been too early to verify a significant drop of CFA in the trial in Orissa. It is difficult to draw a comparison regarding antigenemia with trials not using doxycycline but DEC alone or in combination with ivermectin and/or albendazole, because in a number of clinical trials, the qualitative immuno-chromatography test (ICT) was used to determine antigen positivity. 20,21,28,29 In general, the TropBio is more sensitive; therefore, it is able to detect lower antigenemia levels, causing patients to appear positive for a longer time. 30

Amelioration of dilated scrotal lymphatic vessels.

The degree of dilation in the scrotal lymphatic vessels caused by the adult worms is considered an indirect measure of altered lymphatic function. 31 Vessel dilation is not limited to the immediate vicinity of the worm nest and can be seen in portions of the vessel with no evidence of adult worms. 32 This fact led to the development of a staging system based on measuring the diameter of the largest “empty” vessel. 9,15 In this study, which occurred earlier than the two cited, USG was used to measure the vessel dilation at the sites of worm nests.

The results of this trial showed a significant ameliorating effect on already existent pathology of the scrotal lymphatics in the 21-day doxycycline group (P < 0.0001). This confirms earlier results of a clinical trial conducted by our group in Ghana where the regression of dilated scrotal lymphatics became evident after 12 months.9 In contrast to the 21-day doxycycline group, in this study, Wilcoxon analysis of the endpoint reduction in 10-day and placebo groups was not significant (Table 2). Nevertheless, because all parameters examined here for 10-day doxycycline did show a reduction in some but not all patients, future studies should examine 14-day doxycycline in combination with DEC. Taken together, these results show that DEC does not lead to significant amelioration of LF pathology on its own, a result that was also seen with a 12-day course of DEC treatment. 33 Rather than inducing pathology, as has been seen after DEC (e.g., transient hydroceles), 33 a 21-day course of doxycycline followed by a single dose of DEC improved the patient’s physical condition.

In conclusion, this study showed that a 21-day course of doxycycline (200 mg/day) before administering standard anti-filarial therapy reduces Wolbachia in W. bancrofti by > 90%. After single-dose DEC (6 mg/kg), this combination treatment results in sustained amicrofilaremia, a > 95% reduction in the total number of worm nests, and the loss of FDS in > 90% of the patients. This efficacy is superior to single-dose DEC, and compared with the available literature, also to a 7-day DEC course. The combination of doxycycline and DEC, in contrast to DEC alone, also reverses dilation of lymph vessels and thus improves lymphatic pathology, similar to what was observed for the combination of doxycycline and ivermectin/albendazole in earlier studies. 9,17 It may be this ameliorating effect on pathology that makes the use of doxycycline preferable for the treatment of individuals by doctors, who are charged with selecting a treatment that improves their patients’ disease conditions.

Table 1

Microfilaremia and antigenemia

Table 1
Table 2

Filaria dance sign and total number of worm nests and scrotal lymphatic vessel dilation

Table 2
Table 3

Number of patients who reported adverse reactions 24 hours and 7 days after DEC intake

Table 3
Figure 1.
Figure 1.

Trial profile of patients examined, randomized, and treated. Of the 57 randomized participants, 43 completed the treatment. One patient was absent for USG examination 7 days after DEC (doxycycline 21 days). At the 12-month follow-up, 6 patients were not present for USG examination (doxycycline 21 days, N = 2; doxycycline 10 days, N = 2; placebo, N = 2) and 15 patients refused to give a blood sample (doxycycline 21 days, N = 8; doxycycline 10 days, N = 3; placebo, N = 4).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 4; 10.4269/ajtmh.2009.09-0155

Figure 2.
Figure 2.

Representative image of the left supratesticular area. In the two-dimensional mode (inset: lymph vessel with worm nest is circled), the vertical line indicates the slice shown in the one-dimensional m-mode (large image), where filarial movement over time can be seen as undulating lines in the lumen of a dilated lymphatic vessel. Tissue around the vessel where the worm nest is located appears as horizontal lines. The white dashed line indicates the electronic caliper used to measure vessel diameter (marked with a dashed rectangle). In this example, vessel dilation measured from inner layer to inner layer of the lymphatic vessel was 0.61 cm.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 4; 10.4269/ajtmh.2009.09-0155

*

Address correspondence to Achim Hoerauf, Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. E-mail: hoerauf@microbiology-bonn.de

Authors’ addresses: Sabine Mand, Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel: 49-228-287-15675, Fax: 49-228-287-19573, E-mail: mand@microbiology-bonn.de. Kenneth Pfarr, Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel: 49-228-287-15675, Fax: 49-228-287-19573, E-mail: pfarr@microbiology-bonn.de. Prakash K. Sahoo, Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar 751023, India, Tel: 91-674-2301900 or 2302746, Fax: 91-674-2300728. Ashok K. Satapathy, Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar 751023, India, Tel: 91-674-2301900 or 2302746, Fax: 91-674-2300728. Sabine Specht, Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel: 49-228-287-15675, Fax: 49-228-287-19573, E-mail: specht@microbiology-bonn.de. Ute Klarmann, Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel: 49-228-287-15675, Fax: 49-228-287-19573, E-mail: klarmann@yahoo.de. Alexander Y. Debrah, Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) and Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana, Tel: 233-51-60351, Fax: 233-51-62017, E-mail: yadebrah@yahoo.com. Balachandran Ravindran, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India, Tel: 91-674-2301900 or 2302746, Fax: 91-674-2300728, E-mail: ravindran8@gmail.com. Achim Hoerauf, Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel: 49-228-287-15675, Fax: 49-228-287-19573, E-mail: hoerauf@microbiology-bonn.de.

Acknowledgments: The authors thank the study participants for their cooperation, and Dr. Sidhard Padhi, Bhubaneswar, for assistance with patient care in the villages.

Financial support: We are grateful for financial support from the German Federal Ministry for Education and Research (BMBF), Berlin (Grant IND 02/010), and the Indian Council of Medical Research (ICMR). A.Y.D. was a recipient of scholarship from the German Academic Exchange Service (DAAD) for his PhD work.

REFERENCES

  • 1

    WHO, 2007. Global programme to eliminate lymphatic filariasis. Wkly Epidemiol Rec 82 :361–380.

  • 2

    Dreyer G, Addiss D, Williamson J, Noroes J, 2006. Efficacy of co-administered diethylcarbamazine and albendazole against adult Wuchereria bancrofti. Trans R Soc Trop Med Hyg 100 :1118–1125.

    • Search Google Scholar
    • Export Citation
  • 3

    Hussein O, Setouhy ME, Ahmed ES, Kandil AM, Ramzy RM, Helmy H, Weil GJ, 2004. Duplex Doppler sonographic assessment of the effects of diethylcarbamazine and albendazole therapy on adult filarial worms and adjacent host tissues in bancroftian filariasis. Am J Trop Med Hyg 71 :471–477.

    • Search Google Scholar
    • Export Citation
  • 4

    Ottesen EA, Duke BO, Karam M, Behbehani K, 1997. Strategies and tools for the control/elimination of lymphatic filariasis. Bull World Health Organ 75 :491–503.

    • Search Google Scholar
    • Export Citation
  • 5

    WHO, 2004. Towards a strategic plan for research to support the global program to eliminate lymphatic filariasis. Summary of immediate needs and opportunities for research on lymphatic filariasis, Philadelphia, PA, December 9–10, 2003. Am J Trop Med Hyg 71 :1–46.

    • Search Google Scholar
    • Export Citation
  • 6

    Hoerauf A, Mand S, Adjei O, Fleischer B, Büttner DW, 2001. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357 :1415–1416.

    • Search Google Scholar
    • Export Citation
  • 7

    Hoerauf A, Volkmann L, Hamelmann C, Adjei O, Autenrieth IB, Fleischer B, Büttner DW, 2000. Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet 355 :1242–1243.

    • Search Google Scholar
    • Export Citation
  • 8

    Debrah A, Mand S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Büttner M, Adjei O, Büttner DW, Hoerauf A, 2007. Macrofilaricidal activity after four weeks doxycycline treatment of Wuchereria bancrofti. Trop Med Int Health 12 :1433–1441.

    • Search Google Scholar
    • Export Citation
  • 9

    Debrah AY, Mand S, Specht S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Larbi J, Lawson B, Taylor M, Adjei O, Hoerauf A, 2006. Doxycycline reduces plasma VEGF-C/sVEGFR-3 and improves pathology in lymphatic filariasis. PLoS Pathog 2 :e92.

    • Search Google Scholar
    • Export Citation
  • 10

    Turner JD, Mand S, Debrah AY, Muehlfeld J, Pfarr K, McGarry HF, Adjei O, Taylor MJ, Hoerauf A, 2006. 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 42 :1081–1089.

    • Search Google Scholar
    • Export Citation
  • 11

    Hoerauf A, Mand S, Fischer K, Kruppa T, Marfo-Debrekyei Y, Debrah AY, Pfarr KM, Adjei O, Büttner DW, 2003. Doxycycline as a novel strategy against bancroftian filariasis-depletion of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilaria production. Med Microbiol Immunol (Berl) 192 :261–273.

    • Search Google Scholar
    • Export Citation
  • 12

    Dreyer G, Amaral F, Noroes J, Medeiros Z, Addiss D, 1995. A new tool to assess the adulticidal efficacy in vivo of antifilarial drugs for bancroftian filariasis. Trans R Soc Trop Med Hyg 89 :225–226.

    • Search Google Scholar
    • Export Citation
  • 13

    Faris R, Hussain O, El Setouhy M, Ramzy RM, Weil GJ, 1998. Bancroftian filariasis in Egypt: visualization of adult worms and subclinical lymphatic pathology by scrotal ultrasound. Am J Trop Med Hyg 59 :864–867.

    • Search Google Scholar
    • Export Citation
  • 14

    Mand S, Marfo-Debrekyei Y, Dittrich M, Fischer K, Adjei O, Hoerauf A, 2003. Animated documentation of the filaria dance sign (FDS) in bancroftian filariasis. Filaria J 2 :3.

    • Search Google Scholar
    • Export Citation
  • 15

    Debrah A, Mand S, Toliat MR, Marfo-Debrekyei Y, Batsa L, Nurnberg P, Lawson B, Adjei O, Hoerauf A, Pfarr K, 2007. Plasma vascular endothelial growth factor-A (VEGF-A) and VEGF-A gene polymorphism are associated with hydrocele development in lymphatic filariasis. Am J Trop Med Hyg 77 :601–608.

    • Search Google Scholar
    • Export Citation
  • 16

    Hoerauf A, 2008. Filariasis: new drugs and new opportunities for lymphatic filariasis and onchocerciasis. Curr Opin Infect Dis 21 :673–681.

    • Search Google Scholar
    • Export Citation
  • 17

    Debrah AY, Mand S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Lawson B, Taylor M, Adjei O, Hoerauf A, 2009. Reduction in levels of plasma vascular endothelial growth factor-A and improvement in hydrocele patients by targeting endosymbiotic Wolbachia sp. in Wuchereria bancrofti with doxycycline. Am J Trop Med Hyg 80 :956–963.

    • Search Google Scholar
    • Export Citation
  • 18

    Taylor MJ, Makunde WH, McGarry HF, Turner JD, Mand S, Hoerauf A, 2005. Macrofilaricidal activity following doxycycline treatment of Wuchereria bancrofti: a double-blind randomised controlled trial. Lancet 365 :2116–2121.

    • Search Google Scholar
    • Export Citation
  • 19

    Kazura J, Greenberg J, Perry R, Weil G, Day K, Alpers M, 1993. Comparison of single-dose diethylcarbamazine and ivermectin for treatment of bancroftian filariasis in Papua New Guinea. Am J Trop Med Hyg 49 :804–811.

    • Search Google Scholar
    • Export Citation
  • 20

    El Setouhy M, Ramzy RM, Ahmed ES, Kandil AM, Hussain O, Farid HA, Helmy H, Weil GJ, 2004. A randomized clinical trial comparing single- and multi-dose combination therapy with diethylcarbamazine and albendazole for treatment of bancroftian filariasis. Am J Trop Med Hyg 70 :191–196.

    • Search Google Scholar
    • Export Citation
  • 21

    Kshirsagar NA, Gogtay NJ, Garg BS, Deshmukh PR, Rajgor DD, Kadam VS, Kirodian BG, Ingole NS, Mehendale AM, Fleckenstein L, Karbwang J, Lazdins-Helds JK, 2004. Safety, tolerability, efficacy and plasma concentrations of diethylcarbamazine and albendazole co-administration in a field study in an area endemic for lymphatic filariasis in India. Trans R Soc Trop Med Hyg 98 :205–217.

    • Search Google Scholar
    • Export Citation
  • 22

    Sunish IP, Rajendran R, Mani TR, Dash AP, Tyagi BK, 2006. Evidence for the use of albendazole for the elimination of lymphatic filariasis. Lancet Infect Dis 6 :125–126.

    • Search Google Scholar
    • Export Citation
  • 23

    Hoerauf A, Specht S, Buttner M, Pfarr K, Mand S, Fimmers R, Marfo-Debrekyei Y, Konadu P, Debrah AY, Bandi C, Brattig N, Albers A, Larbi J, Batsa L, Adjei O, Buttner DW, 2008. Wolbachia endobacteria depletion by doxycycline as antifilarial therapy has macrofilaricidal activity in onchocerciasis: a randomized placebo-controlled study. Med Microbiol Immunol (Berl) 197 :295–311.

    • Search Google Scholar
    • Export Citation
  • 24

    Santos MD, Vermeersch H, Remon JP, Schelkens M, De Backer P, Van Bree HJ, Ducatelle R, Haesebrouck F, 1996. Pharmacokinetics and bioavailability of doxycycline in turkeys. J Vet Pharmacol Ther 19 :274–280.

    • Search Google Scholar
    • Export Citation
  • 25

    Noroes J, Dreyer G, Santos A, Mendes VG, Medeiros Z, Addiss D, 1997. Assessment of the efficacy of diethylcarbamazine on adult Wuchereria bancrofti in vivo. Trans R Soc Trop Med Hyg 91 :78–81.

    • Search Google Scholar
    • Export Citation
  • 26

    Weil GJ, Ramzy RM, El Setouhy M, Kandil AM, Ahmed ES, Faris R, 1999. A longitudinal study of bancroftian filariasis in the Nile Delta of Egypt: baseline data and one-year follow-up. Am J Trop Med Hyg 61 :53–58.

    • Search Google Scholar
    • Export Citation
  • 27

    Mand S, Debrah A, Batsa L, Adjei O, Hoerauf A, 2004. Reliable and frequent detection of adult Wuchereria bancrofti in Ghanaian women by ultrasonography. Trop Med Int Health 9 :1111–1114.

    • Search Google Scholar
    • Export Citation
  • 28

    Helmy H, Weil GJ, Ellethy AS, Ahmed ES, Setouhy ME, Ramzy RM, 2006. Bancroftian filariasis: effect of repeated treatment with diethylcarbamazine and albendazole on microfilaraemia, antigenaemia and antifilarial antibodies. Trans R Soc Trop Med Hyg 100 :656–662.

    • Search Google Scholar
    • Export Citation
  • 29

    Meyrowitsch DW, Simonsen PE, Magesa SM, 2004. Long-term effect of three different strategies for mass diethylcarbamazine administration in bancroftian filariasis: follow-up at 10 years after treatment. Trans R Soc Trop Med Hyg 98 :627–634.

    • Search Google Scholar
    • Export Citation
  • 30

    Njenga SM, Wamae CN, Njomo DW, Mwandawiro CS, Molyneux DH, 2008. Impact of two rounds of mass treatment with diethylcarbamazine plus albendazole on Wuchereria bancrofti infection and the sensitivity of immunochromatographic test in Malindi, Kenya. Trans R Soc Trop Med Hyg 102 :1017–1024.

    • Search Google Scholar
    • Export Citation
  • 31

    Peixoto CA, Figueiredo-Silva J, 2001. Fine structure of intrascrotal lymphatic vessels infected by Wuchereria bancrofti adult worms. J Submicrosc Cytol Pathol 33 :125–131.

    • Search Google Scholar
    • Export Citation
  • 32

    Dreyer G, Noroes J, Figueredo-Silva J, Piessens WF, 2000. Pathogenesis of lymphatic disease in bancroftian filariasis: a clinical perspective. Parasitol Today 16 :544–548.

    • Search Google Scholar
    • Export Citation
  • 33

    Freedman DO, Bui T, De Almeida Filho PJ, Braga C, Maia e Silva MC, Maciel A, Furtado AF, 1995. Lymphoscintigraphic assessment of the effect of diethylcarbamazine treatment on lymphatic damage in human bancroftian filariasis. Am J Trop Med Hyg 52 :258–261.

    • Search Google Scholar
    • Export Citation
Save