Volume 7, Number 7—June 2001
Doxycycline and Eradication of Microfilaremia in Patients with Loiasis
To the Editor: Wolbachia are intracellular symbionts found in 20% of insects and in several nematodes, including filarial worms. Because tetracycline eradicates Wolbachia in nematodes, this drug has been proposed for chemotherapy in filariasis (1). We report two patients with loiasis in whom no Wolbachial DNA was detected in microfilariae by polymerase chain reaction (PCR), and for whom 6 weeks of doxycycline failed to eradicate the microfilaremia. We conclude that doxycycline may not be an efficient therapy for loiasis.
Filariae are responsible of 150 million infections worldwide, some of them devastating diseases such as elephantiasis (caused by Bancroftian and Brugian filariasis) and blindness (caused by onchocerciasis). There is no satisfactory treatment for filariasis: although diethycarbamazine citrate has been used for 50 years to treat the disease, this drug is not efficient in most adults (2). Ivermectin has been reported to be efficient in treating microfilaremia and possibly for viability and fertility of adult Oncocerca volvulus worms if therapy is prolonged for 2 to 3 years (2). However, the microfilaricidal effect of ivermectine on Wuchereria, Brugia, and Loa loa is similar to diethycarbamazine: ultrasonography shows that it has no effect on adult Bancroftian worms, and microfilariae often reappear after a few months (2).
Intracellular bacteria have been observed in the lateral cords of adult female worms as well as in microfilariae of W. bancrofti, and these bacteria have recently been identified as belonging to the Wolbachia genogroup (3). Wolbachia have been detected by PCR in Brugian and Bancroftian filariae, dirofilariae, and most species of Oncocerca including O. volvulus, but have never been detected in L. loa worms (4). Wolbachia are causative agents of a variety of modifications in host development and reproduction, including cytoplasmic incompatibility and parthenogenesis. Consequently, it has been proposed that antibiotic eradication of Wolbachia from infected filarial worms would reduce microfilaremia. This has been demonstrated in an animal model with Litomosoides sigmodontis and recently confirmed in patients infected with O. volvulus (1-5). In experimental L. sigmodontis infection, after 41 days of therapy, microfilaremia in tetracycline-treated animals was one-tenth that in normally infected animals (5).
We report the failure of tetracycline to reduce microfilaremia in two patients with L. loa filariasis. Patient no. 1 was a 58-year-old man who worked in Gabon for many years and had cutaneous larva migrans. L. loa microfilaremia was detected (4x103/mL). Patient no. 2, a 15-year-old boy living in Cameroon, was diagnosed with Calabar swelling with L. loa microfilaremia (1x103/mL). After giving informed consent, both patients were treated with doxycycline 200 mg daily for 6 weeks as previously described in O. volvulus-infected patients (1). We observed patients for microfilaremia every week for 6 weeks and then every 2 weeks for 2 months. The presence of adult worms was detected by physical examinations.
Microfilaremia was detected in both patients at the completion of treatment and at day 120 of followup. In patient no. 1, the frequency of migrating adult worms seemed to diminish during therapy, but they never disappeared. For Wolbachia detection in worms, blood samples were collected both in Dupont-Isolator and EDTA-containing tubes. After centrifugation at 5000 x g for 30 min, the worm-enriched pellet was resuspended in 1 mL of sterile deionized water for erythrocyte lysis. DNA was extracted from the suspension by using the QIAmp-blood kit (Qiagen, Hilden, Germany) following manufacturer's recommendations. W. pipientis DNA was used as a positive control. Control of DNA extraction was performed by amplifying microfilarial DNA using the nematode-specific 18S rDNA-derived primers 18SF (5'-GAT-ACC-GCC-CTA-GTT-CTG-ACC-3') and 18SR (5'-ACC-AAC-TAA-GAA-CGG-CCA-TG-3'). Wolbachia detection was attempted with the FD1 (5'-AGA-GTT-TGA-TCC-TGG-CTC-AG-3') and Rp2 (5'-ACG-GCT-ACC-TTG-TTA-CGA-CTT-3') eubacterial primers, with the Ehrlichia genus-specific 16S r DNA primers EHR16SD (5'-GGT-ACC-YAC-AGA-AGA-AGT-CC-3') and EHR16SR (5'- TAG-CAC-TCA-TCG-TTT-ACA-GC-3'), and with primers specific for the 16S r DNA of B. malayi endosymbiont, Bsymbf (5'-ACG-AGT-TAT-AGT-ATA-ACT-3'), and Bsymbr (5'-CCT-TCG-AAT-AGG-AAT-AAT-3') (3-6). PCR reactions were performed on PTC-200 thermocycler (MJ-Research, USA) by using 45 cycles of denaturation at 94°C for 30 sec, hybridization for 45 sec, and elongation at 72°C for 1 min. Hybridization temperatures were 55°C for FD1/Rp2, 53°C for EHR16SD/ EHR16SR, 42°C for Bsymbf/Bsymbr, and 57°C for 18SF/18SR. Experiments were repeated three times.
We detected Wolbachia in the positive control and 18s rRNA of the nematode in the sample, but no signal compatible with Wolbachial DNA was obtained with the sets of primers used. In fact, four species of filariae (Dipetalonema setariosum, Acanthocheilonema vitae, O. flexuosa, and L. loa) tested for intracellular bacteria by electron microscopy, immunohistochemistry, or PCR had no bacteria (4). The absence of Wolbachia in L. loa microfilariae may explain the failure of tetracycline therapy in our patients.
More work is needed to determine the prevalence of Wolbachia in filariae, their impact on fertility in each species, and the use of antibacterial agents for eradicating these pathogens.
We thank M.J. Taylor for providing W. pipientis DNA for controls.
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