Tropical Medicine and International Health volume 6 no 1 pp 37±41 january 2001 Effect of aggressive prolonged diethylcarbamazine therapy on circulating antigen levels in bancroftian lariasis David O. Freedman 1, D. Adam Plier 1, Adriana B. de Almeida 1, Ana Luna de Oliveira 1, Janaina Miranda 2 and Cynthia Braga 2 1 Division of Geographic Medicine, Department of Medicine, University of Alabama, Birmingham, USA 2 Centro de Pesquisas Aggeu MagalhaÄes, FundacËaÄo Oswaldo Cruz, Recife, Pernambuco, Brazil Summary BACKGROUND Single dose diethylcarbamazine (DEC) as used in control programmes is effectively micro laricidal for periods of up to a year or more but has incomplete ability to kill Wuchereria bancrofti adult parasites. These regimens can be effective in breaking transmission by suppression of circulating micro lariae available to mosquito vectors. Whether prolonged or aggressive therapy with DEC has a signi cant effect on adult worms, which may live up to 12 years or more, and is important in the context of the treatment of individual patients, is still incompletely understood. METHODS In order to investigate the adulticidal effect of aggressive therapy, DEC was given at 6 mg/kg/day for 12-day courses at 0, 6, 12, and 18 months and Og4C3 antigenaemia followed over two years in 38 CAg + Brazilians in a W. bancrofti endemic area. RESULTS At two year follow-up, the median level of antigenaemia was 21% of the pre-treatment value. 92% of individuals had antigen levels < 50% of pretreatment values, but only 26% had completely cleared antigenaemia. The clearance rate at 24 months was only 12% (3/26) in the asymptomatic CAg + patients but 58% (7/12) in those with clinical manifestations of lariasis. The latter individuals cleared signi cantly more antigen (median of 0% pretreatment antigenaemia vs. 26%; P ˆ 0.02) than asymptomatic but infected individuals. CONCLUSION Aggressive repeated therapy with DEC alone is ineffective in consistently eradicating adult W. bancrofti, especially in infected but asymptomatic individuals. Prolonged courses of combination therapy with other anti larial drugs should be investigated for treatment of individual patients with the means to pursue aggressive personal medical care. keywords W. bancrofti, DEC, antigenaemia, control programmes correspondence David O. Freedman, Division of Geographic Medicine, Department of Medicine, University of Alabama at Birmingham, 1530 3rd Ave S., BBRB #203, Birmingham, AL 35294-2170, USA. Telephone: 00 1 205 934 1630; Fax: 00 1 205 934 5600; E-mail: dfreedman@geomed.dom.uab.edu Introduction More than 120 million people in 76 countries are infected with the lymphatic larial parasites Wuchereria bancrofti (90% of cases) and Brugia malayi (10% of cases). Single doses or single short courses of diethylcarbamazine (DEC), ivermectin, or albendazole either alone or in combination are effectively micro laricidal for periods of up to a year or more. These regimens are designed for public health programs where the aim is to break transmission by suppression of circulating micro lariae available to mosquito vectors. However, the effect of any of these drugs in killing adult Wuchereria bancrofti worms, which may live for 12 years or more, is still incompletely understood. With the recent ability to measure serum circulating antigen (CAg), a sensitive indicator of the presence of live adult larial parasites, some direct data on the dosage and time required for DEC to kill adult worms is starting to emerge. In studies of single dose or single course regimens of up to 72 mg/kg total dose, antigenaemia is completely cleared in only 0±18% of infected individuals followed for one to three years (Weil et al. 1988, 1999; Eberhard et al. 1997; ã 2001 Blackwell Science Ltd 37
Nicolas et al. 1997; Ismail et al. 1998). In these studies, at follow-up, median pretreatment antigen levels ranged from 23% to 57% with extremely wide variations in antigen clearance among individuals within each patient cohort. As DEC has clear but incomplete adulticidal activity in limited courses, the question of whether prolonged or aggressive therapy with DEC has more signi cant adulticidal activity is important in the context of individual patients. In asymptomatic infected individuals (either from endemic areas or from other countries) with suf cient means for personal medical care, eradication of adult parasites might prevent onset of clinical disease, or attenuate disease in those who already have clinical manifestations of lymphatic in ammation. We wanted to assess, by following circulating antigenaemia over two years, the adulticidal effect of aggressive therapy with DEC given at 6 mg/kg/day for 12-day courses repeated every six months. Materials and methods Human subjects We studied 38 subjects residing in neighbouring communities of metropolitan Recife, Brazil, where W. bancrofti is endemic, from June 1997 to July 2000. Subjects were initially classi ed into two groups based on their current infection status (CAg+ vs. CAg±). All subjects, regardless of CAg status or clinical manifestations, were treated with diethylcarbamazine (DEC) 6 mg/kg/day in three divided doses for 12 days and were then treated again at 6, 12, and 18 months using the same dosing regimen. Prior to each treatment course, adherence to the previous course was veri ed by pill counts and detailed one-to-one private interviews, and a daytime serum for CAg was drawn and frozen at )70 C. Patients missing one or more treatment courses were excluded from nal analysis. Subjects were otherwise free of any intercurrent illness. None had received diethylcarbamazine, albendazole, or ivermectin therapy within the previous ve years. out by the manufacturer (Trop-Ag W. bancrofti, TropBio Pty Ltd, Townsville, Australia). Boiling pre-treatment was used and the nal serum dilution was 1:4. Samples were run in duplicate. Serial specimens on individual patients were run in adjacent wells. Results are expressed in manufacturer's arbitrary antigen units using the supplied Onchocerca gibsoni standards (negative cutoff ˆ 32 units). The assay does not cross-react with other larial species or related intestinal nematodes (More & Copeman 1990; Weil et al. 1997). The larial antigen test detects adult worm products in human sera and a positive result indicates the presence of living adult worms. Statistical analysis Data management and analyses were performed with Statview version 4.0 software. The non-parametric Mann± Whitney U-test was used for comparison of ages and antigen clearance between patient groups. Results Patient population As shown in Table 1, at enrolment, 38 subjects were CAg+ with a median age of 30.5 years (range 15±62). 22 were male (median age 30.0 years) and 16 female (median age 30.5 years). 26/38 were asymptomatic (clinical lariasis negative, CF±) with no current or previous history of adenolymphangitis, erysipelas, cellulitis, or limb swelling. 12/38 individuals had clinical lariasis (CF+) including eight males with hydrocele only and four females with lymphoedema. Median age did not differ between CF+ (32 years) and CF± (30.5 years) individuals. Nineteen CAg± control individuals with overt clinical lariasis (with positive anti larial IgG) were treated in the same way and remained CAg± through follow-up, and will not be discussed further. Ethical approval All subjects gave written informed consent according to human experimentation guidelines of the US Department of Health and Human Services. The protocol was approved by the Institutional Review Board of the University of Alabama at Birmingham. Circulating antigen assay Levels of circulating W. bancrofti antigen in sera were determined using the Og4C3 antigen ELISA exactly as set Table 1 Clinical ndings in study subjects Clinical classi cation Number of study subjects Male/ Female Age (range) CAg+ CF± 26 14/12 30.5 (15±62) CAg+ CF+ (total) 12 8/4 32.0 (15±54) CAg+ CF+ hydrocele 8 8/0 25.5 (15±42) CAg+ CF+ lymphoedema 4 0/4 49.0 (35±54) CAg±CF+ (controls) 19 4/15 36.0 (24±62) 38 ã 2001 Blackwell Science Ltd
Filarial antigen clearance Figure 1 shows individual antigen unit levels for all individual CAg + patients at 0, 12, and 24 months. While every patient had decreasing antigen levels with time as a result of the repeated aggressive treatment, only 5.7% (2/35) completely cleared antigenaemia after one treatment (6-month follow-up; data not shown) and only 26% (10/38) completely cleared after two years. Interestingly, the clearance rate at 24 months was only 12% (3/26) in the asymptomatic CAg+ CF± patients but 58% (7/12) in the CAg+ CF+ individuals. 38% (3/8) of CF + patients with hydrocele only as compared to 100% (4/4) with lymphoedema completely cleared antigenaemia. In order to quantify larial antigen clearance with time, antigen unit levels were examined as a percentage of each CAg+ individual's pre-treatment value (Fig. 2a). As clearance of antigen was highly variable between individuals, median values are used to compare time points. After one course of DEC (6-month follow-up) the median (25±75% interquartile range) level of CAg was 53% (35±73%) of the pre-treatment value. This decreased with further regular courses of DEC to 32% (19±55%) at 12 months and 21% (4±36%) at 24 months. At 24 months, 92% (35/38) of individuals had antigen levels that were less than 50% of pre-treatment values and the other three individuals had at least a moderate response (78, 71, 71% of pre-treatment). In marked contrast, after one course of DEC, only 43% (15/35) had antigen levels < 50% of pre-treatment values. Figure 1 Filarial antigen levels expressed in Og4r C3 antigen units in individual CAg+ patients after two courses of DEC 6 mg/ kg/day for 12 days (12-month follow-up) and after six-monthly therapy for two years. Open circles indicate asymptomatic (CF±) individuals and closed circles individuals with clinical lariasis (CF+). Negative cutoff value for the assay is 32 units. Figure 2(a) kinetics of larial antigen clearance over time in all 38 CAg+ individuals. Antigen levels are expressed as a percentage of each individuals pretreatment antigen value. (b) comparison of antigen clearances at each time point between asymptomatic (CF±) and clinical lariasis (CF+) patients. For each time point the boxes and horizontal line delimit the 25th and 75th percentile as well as the median pretreatment value. The whiskers denote 10th and 90th percentiles and outlying points are each separately shown. ã 2001 Blackwell Science Ltd 39
Pre-treatment antigen unit values were not signi cantly different between CAg+ CF+ (geometric mean ˆ 820 units) and asymptomatic CAg+ CF± (geometric mean ˆ 874 units) individuals. However, when median antigen clearances were compared between the CF+ and CF± groups (Fig. 2b), we found that the CF+ group cleared signi cantly more antigen at 6 months (CF+ ˆ 33% pretreatment vs. CF± ˆ 58%; P ˆ 0.02), 12 months (CF+ ˆ 17% pre-treatment vs. CF± ˆ 38%; P ˆ 0.008), and 24 months (CF+ ˆ 0% pre-treatment vs. CF± ˆ 26%; P ˆ 0.02). Discussion The degree of activity of the three major anti larial drugs (DEC, ivermectin, albendazole) against adult Wuchereria bancrofti worms is still incompletely understood. With the recent ability to measure serum CAg, a sensitive indicator of the presence of live adult larial parasites, some direct data on the dosage and time required for these drugs to kill adult worms is emerging. Animal studies have shown that CAg levels correlate with the number of adult worms in the host. Decreased CAg after treatment in humans should thus re ect proportionate death of adult worms. Most published studies have followed CAg levels over time after single dose or single course (up to 72 mg/kg) therapies (Weil et al. 1988; Eberhard et al. 1997; Nicolas et al. 1997; Ismail et al. 1998; Weil et al. 1999) used in the context of control programs where the primary goal has been to reduce micro laraemia and interrupt transmission to mosquito vectors. In Tahiti, after a 6-mg/kg single dose of DEC, the median level of CAg was 29.6% of pretreatment values at two years and 9.5% of treated individuals completely cleared antigenaemia (Nicolas et al. 1997). In Egypt, DEC 6 mg/kg/day for 12 consecutive days resulted in median levels of CAg of 57% pre-treatment values and a complete clearance rate of 18% at 1 year (Weil et al. 1999). However, in that study, an untreated group had a complete clearance rate of 20%. In Haiti, DEC 6 mg/kg once per week for 12 weeks resulted in a median level of CAg 25% of pre-treatment values at three years with no patient completely clearing antigenaemia (Eberhard et al. 1997). In Sri Lanka, DEC 6 mg/kg in a single dose combined with a single dose of 600 mg of albendazole resulted in median pre-treatment levels of 23% at 15 months with no patient completely clearing antigenaemia (Ismail et al. 1998). This was signi cantly better than in a comparison group treated in the same study with albendazole alone and there was no DEC-only arm. Thus, single course therapy appears to result in no better than a residual antigenaemia of 23% of pretreatment levels. Only two studies have examined CAg levels after slightly more aggressive treatment. Ismail et al. (1996) treated 12 asymptomatic Sri Lankan individuals with 10 mg/kg doses of DEC repeated every two weeks for 11 doses (total dose 110 mg/kg). After two years, 7/12 had completely cleared antigenaemia and the median level of CAg was 10.0% of pre-treatment values. McCarthy et al. (1995) treated 18 Cook Islanders with DEC 8 mg/kg/day for 7 consecutive days repeated ve more times over 6 months (total dose 336 mg/kg) resulting in complete antigen clearance in 80% at 18 months. However, in that study, the subjects were noted to be extremely lightly infected with median micro- laria counts of just 32 mf/ml and with mean CAg levels signi cantly lower than those found in other parts of the South Paci c. We examined a cohort with substantial CAg levels treated repeatedly (every six months) over a two year period (total DEC dose 288 mg/kg). At 24 months, the median level of CAg was 21% of pre-treatment values and the complete clearance rate of 26% is comparable with that found in the studies discussed above. Ultrasonographic visualization of scrotal lymphatics after treatment has suggested inter-individual variability in susceptibility of adult W. bancrofti to DEC (Noroes et al. 1997). The present study extends these preliminary ndings on localized adult worms by examining the response to DEC of the total body burden of adult W. bancrofti as manifest by CAg levels. An unexpected nding, however, was the striking and signi cantly increased adulticidal effect of DEC in those CAg+ individuals who also had overt clinical manifestations of lariasis (Figs 1 and 2b). This was manifest by an overall clearance rate of 58% vs. 12% in CF + compared to CF± subjects and a median residual antigenaemia of 0% vs. 26% in the former compared to the latter group. Lymphoedema patients are generally older and thought to be at a more advanced stage of disease than those with hydrocele only. 38% (3/8) of CF+ patients with hydrocele only completely cleared antigenaemia, compared to 100% (4/4) with lymphoedema. Previous work has suggested that these CAg+ CF+ individuals possess clinical and immunological characteristics that distinguish them from those that are CAg+ and asymptomatic, and that they should be treated as a separate group (de Almeida et al. 1996; Freedman 1998). One may hypothesize that the same host factors that lead to increased in ammatory manifestations to the larial infection in these individuals may potentiate the actions of DEC in clearing adult W. bancrofti worms. Though the numbers are small, the nding that all individuals in the group with the most advanced in ammatory disease, the lymphoedema patients, cleared antigenaemia, compared to 40 ã 2001 Blackwell Science Ltd
38% of hydrocele patients and 12% of asymptomatic infected individuals, supports this hypothesis. These overall results indicate: considerable inter-individual heterogeneity in response to DEC indicating a possible contribution of host factors; some bene t of prolonged therapy on residual antigenaemia in CF+ patients but only a very modest effect in CF± individuals; and an inability to eradicate adult larial worms in most individuals even with prolonged DEC therapy. Combination therapy with more than one anti larial drug is now recommended for short course regimens in control programs. Prolonged courses of DEC are currently recommended as the therapy of choice for individual patients with clinical manifestations of bancroftian lariasis. Our results indicate that this is wholly inadequate for eradicating all viable adult parasites in the asymptomatic individuals that comprise the bulk of most infected populations. Prolonged courses of combination therapy should be investigated for treatment of individual patients with the nancial means for personal medical care, where the goal should be to eradicate adult parasites. Acknowledgements We thank Drs Alexandre Bezerra de Carvalho and Andre Furtado for the continuing support of CPqAM in conducting these studies, and are grateful to Luiz Carlos Alves and LaÃnia Ferreira da Silva for technical and data assistance in Brazil. We thank the staff of the Secretary of Health, Municipality of Olinda for use of clinical facilities and for logistical support. Financial support was provided by PHS grant NIAID RO1 AI 31552. References Eberhard ML, Hightower AW, Addiss DG & Lammie PJ (1997) Clearance of Wuchereria bancrofti antigen after treatment with diethylcarbamazine or ivermectin. American Journal of Tropical Medicine and Hygiene 57, 483±486. Freedman DO (1998) Immune dynamics in the pathogenesis of human lymphatic lariasis. Parasitology Today 14, 229±234. Ismail MM, Jayakody RL, Weil GJ et al. (1998) Ef cacy of single dose combinations of albendazole, ivermectin and diethylcarbamazine for the treatment of bancroftian lariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 94±97. Ismail MM, Weil GJ, Jayasinghe KSA et al. (1996) Prolonged clearance of micro laremia in patients with bancroftian lariasis after multiple high doses of ivermectin or diethylcarbamazine. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 684±688. McCarthy JS, Guinea A, Weil GJ & Ottesen EA (1995) Clearance of circulating larial antigen as a measure of the macro laricidal activity of diethylcarbamazine in Wuchereria bancrofti infection. Journal of Infectious Diseases 172, 521±526. More SJ & Copeman DB (1990) A highly speci c and sensitive monoclonal antibody-based ELISA for the detection of circulating antigen in bancroftian lariasis. Tropical Medicine and Parasitology 41, 403±406. Nicolas L, Plichart C, Nguyen LN & Moulia Pelat JP (1997) Reduction of Wuchereria bancrofti adult worm circulating antigen after annual treatments of diethylcarbamazine combined with ivermectin in French Polynesia. Journal of Infectious Diseases 175, 489±492. NoroÄ es J, Dreyer G, Santos A, Mendes VG, Medeiros Z & Addiss D (1997) Assessment of the ef cacy of diethylcarbamazine on adult Wuchereria bancrofti in vivo. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 78±81. Weil GJ, Lammie PJ & Weiss N (1997) The ICT Filariasis test: a rapid-format antigen test for diagnosis of bancroftian lariasis. Parasitology Today 13, 401±404. Weil GJ, Ramzy RM, El Setouhy M, Kandil AM, Ahmed ES & Faris R (1999) A longitudinal study of Bancroftian lariasis in the Nile Delta of Egypt: baseline data and one-year follow-up. American Journal of Tropical Medicine and Hygiene 61, 53±58. Weil GJ, Sethumadhavan KV, Santhanam S, Jain DC & Ghosh TK (1988) Persistence of parasite antigenaemia following diethylcarbamazine therapy of bancroftian lariasis. American Journal of Tropical Medicine and Hygiene 38, 589±595. ã 2001 Blackwell Science Ltd 41