Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar.

Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar M. Albonico,1 Q. Bickle,2 M. Ramsan,3 A. Montresor,4 L. Savioli,4 & M. Taylor2 Objective To evaluate the efficacy of and resistance to mebendazole (500 mg) and levamisole (40 or 80 mg), alone or in combination, for the treatment of Ascaris lumbricoides, Trichuris trichiura and hookworm infections on Pemba Island — an area exposed to periodic school-based mebendazole treatment since 1994. Methods A randomized, placebo-controlled trial was carried out in 914 children enrolled from the first and fifth grades of primary schools. Stool samples collected at baseline and 21 days after treatment were examined by the Kato–Katz technique to assess the prevalence and intensity of helminth infection. Findings Efficacies of mebendazole and levamisole as single treatments against intestinal nematode infections were comparable with those in previous trials, but mebendazole treatment of hookworm infections gave significantly lower cure (7.6%) and egg reduction (52.1%) rates than reported in a study undertaken before the beginning of periodic chemotherapy (cure rate, 22.4%; egg reduction rate, 82.4%). Combined treatment with mebendazole and levamisole had a significantly higher efficacy against hookworm infections (cure rate, 26.1%; egg reduction rate, 88.7%) than either drug given alone. No difference in mebendazole efficacy was found in children who had been treated repeatedly compared with those who had not been treated previously. Conclusion The overall efficacy of mebendazole against hookworm infections after periodic chemotherapy is reduced. The efficacy of benzimidazoles in chemotherapy-based control programmes should be monitored closely. Combined treatment with mebendazole and levamisole may be useful as a tool to delay the development of benzimidazole resistance. Keywords Ascariasis/drug therapy; /Trichuriasis/drug therapy; Ascaris lumbricoides/drug effects; Trichuris/drug effects; Mebendazole/ pharmacology/administration and dosage; Levamisole/pharmacology/administration and dosage; Placebos; Drug therapy, Combination; Treatment outcome; Ancylostoma/drug effects; Necator americanus/drug effects; Drug resistance; Randomized controlled trials; Comparative study; United Republic of Tanzania (source: MeSH, NLM ). Mots cle´s Ascaridiase larvaire/chimiothe´rapie; Trichoce´phalose/chimiothe´rapie; Ascaris lumbricoides/action des produits chimiques; Trichuris/action des produits chimiques; Me´bendazole/pharmacologie/administration et posologie; Le´vamisole/pharmacologie/ administration et posologie; Placebo; Polychimiothe´rapie; Evaluation re´sultats traitement; Ankylostome/action des produits chimiques; Necator americanus/action des produits chimiques; Re´sistance aux me´dicaments; Essai clinique randomise´; Etude comparative; Re´publique-Unie de Tanzanie (source: MeSH, INSERM ). Palabras clave Ascariasis/quimioterapia; Tricuriasis/quimioterapia; Ascaris lumbricoides/efectos de drogas; Trichuris/efectos de drogas; Mebendazol/farmacologı´a/administracio´n y dosificacio´n; Levamisol/farmacologı´a/administracio´n y dosificacio´n; Placebos; Quimioterapia combinada; Resultado del tratamiento; Ancylostoma/efectos de drogas; Necator americanus/efectos de drogas; Resistencia a las drogas; Ensayos controlados aleatorios; Estudio comparativo; Repu´blica Unida de Tanzanı´a (fuente: DeCS, BIREME ). Bulletin of the World Health Organization 2003;81:343-352. Voir page 350 le re´sume´ en franc¸ais. En la pa´gina 350 figura un resumen en espan˜ol. Introduction Public health programmes to controlmorbidity associatedwith soil-transmitted helminth infections depend mainly on the delivery of anthelminthic drugs to primary-school children (1). In theory, four single-dose drugs are available (albendazole, levamisole, mebendazole, and pyrantel); in practice, however, most control programmes only use the benzimidazoles (albendazole and mebendazole) because they are given as a single-dose tablet and children do not need to be weighed (2). 1 Ivo de Carneri Foundation, Milan, Italy. 2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, England. Correspondence should be addressed to Dr Bickle (email: quentin.bickle@lshtm.ac.uk). 3 Public Health Laboratory Ivo de Carneri, Pemba Island, Zanzibar, United Republic of Tanzania. 4 Parasitic Diseases and Vector Control, Communicable Diseases, World Health Organization, Geneva, Switzerland. Ref. No. 02-0263 343Bulletin of the World Health Organization 2003, 81 (5) Aside from reducing the load of worms, benzimidazole treatment also improves the nutritional status and cognitive development of children infected with Ascaris lumbricoides, Trichuris trichiura, and hookworms and reduces hookworm- associated anaemia in children and in women of childbearing age (3–9). A number of studies have shown the short- and long-term benefits of periodic treatment with a single dose of 500 mg mebendazole in endemic areas (8, 10, 11). Levamisole also is used in helminth control programmes with good results, although it is less effective than mebendazole against T. trichiura andNecator americanus infections. Comparative trials of both drugs, alone or in combination, are few, however, and none have compared single-dose administration of 500 mg mebendazole and 40 or 80 mg levamisole (12–16). At present, concern exists about the possible emergence of drug resistance to the anthelminthic compounds used to control intestinal nematodes in humans, particularly given the well-documented and widespread problem of anthelminthic resistance in livestock as a consequence of frequent periodic mass treatments (17). The efficacy of combined treatments that use anthelminthics with differing modes of action (e.g. mebendazole plus levamisole) need to be assessed, both to explore possible additive or synergistic effects and to identify a combination that could delay the occurrence of anthelminthic drug resistance to each class of drug (18). Comparison of the efficacy of mebendazole in the treatment ofA. lumbricoides, T. trichiura, and hookworm infection in an area where the school-age population has been exposed to the drug for the last five years with data collected before the beginning of the control programme andwith other efficacy data collected elsewhere can give valuable information on possible changes in drug susceptibility of worms. The primary purpose of this trial was to evaluate the efficacy of single-dose mebendazole in an area where this drug has been used widely for periodic chemotherapy targeted at schoolchildren. A secondary objective was to assess and compare the efficacy of mebendazole with that of levamisole given as one or two 40-mg tablets and the efficacy of the two drugs given in combination. Materials and methods Study area and study population The study was carried out on Pemba Island, the smaller of the two islands of Zanzibar, United Republic of Tanzania. Important features of the island have been described in detail elsewhere (19, 20). Intestinal helminth infections affect most of the population and cause a heavy burden of disease in children and in women of childbearing age (21). The National HelminthControl Programmewas initiated inZanzibar in June 1994, and since then, mebendazole 500 mg has been given to schoolchildren as a single dose every 4–6 months (21). The study was conducted in August 1999 among children enrolled in the first grade (Standard 1) and fifth grade (Standard 5) of 10 public schools on Pemba Island. The schools were chosen randomly from the 72 schools on the island. Children at Standard 1 had not yet been treated with mebendazole in school, whereas children at Standard 5 had been exposed to 15 rounds of mebendazole treatment. Children were excluded from the trial if they did not have parental or guardian permission to participate, did not provide a stool sample, had significant comorbidities (e.g. severe diarrhoea, severe anaemia, or high fever), or had recently transferred to the school from an area outside Zanzibar. Study design The study was a randomized, placebo-controlled trial. Before children were enrolled in the study, parents or guardians of children in the selected schools were given a comprehensive explanation of the risk and benefits of the trial, and verbal consent was sought. Children enrolled in the study were assigned randomly to one of four treatment groups to receive 500 mg mebendazole (Janssen, Belgium), 40 or 80 mg levamisole (Zeneca, UK), 500 mg mebendazole plus 40 or 80 mg levamisole, or placebo. Placebo pills resembled mebendazole in colour, size, taste, and shape. On the day before the scheduled treatment date, children eligible to participate in the trial were given a container in which to bring a fresh stool sample the next day. On the day of treatment, the stools were collected and the children’s weights were recorded. Randomization was blocked on weight, and a computer-generated programme was used to create two randomized treatment lists: one for children who weighed 15–20 kg, who were to receive one tablet of 40 mg levamisole, and another for children who weighed 21–60 kg, who were to receive two tablets (80mg). One tablet ofmebendazole 500mg and one of placebo was given irrespective of body weight. Treatments given were placed in sealed, opaque envelopes and were coded with a number. Children were identified by these numbers only throughout the study. Twenty-one days after treatment, all children were revisited to collect a further stool sample. Any child who failed to bring a stool sample was followed for up to 24 days. Parents and children were instructed to report to the teacher and refer to the nearest health centre with any severe adverse effects that occurred in the week after treatment. After completion of the study, children in the placebo group and children positive after the follow-up survey were treated with mebendazole 500 mg. The study was approved by the Zanzibar Health Research Council, and by the ethical committees of WHO and the London School of Hygiene and Tropical Medicine. Parasitology After both surveys, egg counts in stool samples were assessed within sixhoursof the samplebeingproducedat thePublicHealth Laboratory Ivo de Carneri. All laboratory investigations were blinded, so the technicianswhoexamined the slideswereunaware of the treatment the patients received. Stools were analysed using theKato–Katz techniqueaccording toWHOguidelines (22).The slides were examined within one hour of preparation to avoid overclarification of hookworm eggs. Comparison of egg counts before and after treatment allowed calculation of the cure rate and the egg reduction rate. A random sample of 10% of the smears prepared for the Kato–Katz technique was read by two different technicians to evaluate the accuracy of the diagnosis and the precision of the egg counts. Slides were re-examined if the quality control showed a >10% difference in egg counts. To describe the egg count distribution in the study population before and after treatment, intensity of infection was classified as ‘‘light’’, ‘‘moderate’’, or ‘‘heavy’’ on the basis of faecal egg counts. As no heavy infections with A. lumbricoides were present at baselinewhen analysed according toWHOcut- off points, categories for A. lumbricoides intensity of moderate and heavy infection were revised to include a meaningful number of children for comparison (Table 1). Categories for intensity of T. trichiura and hookworm infection are according to WHO guidelines (Table 1) (23). 344 Bulletin of the World Health Organization 2003, 81 (5) Research Statistical analysis Data were entered and analysed with EpiInfo software. Cure rates were calculated as the percentage of children with egg counts >0 before treatment who had negative egg counts after treatment. The percentage reduction in prevalence was calculated as [(N+/n) – (N21 +/n)]/(N+/n), where N+ = the number of positive children at baseline, N21 + = the number of positive children 21 days after treatment, and n = the total number of children with samples from both day 0 and day 21. Both cure rates and percentage reductions in prevalence were calculated. As the sensitivity of the Kato–Katz method could be influenced by the intensity of infection, however, reductions in prevalence were comparable only when pre-treatment intensities of infection were similar. The percentage reduction in eggs induced by treatment was estimated as 100[1 – exp(–D)]%], whereD = S (loge(E21) – loge(E0))/n, E21 = the egg count at 21 days (for each individual), E0 = the count before treatment, and n = the number of children. Confidence intervals were based on the inter-individual variation in the differences in the logarithms of the egg counts. Proportions were compared with standard w2 tests. Geometric mean egg counts were estimated as exp[S(loge(c+1))/n] – 1, where c = the count (eggs per gram) for a particular individual and n= the total number of samples. Geometric means were compared with analyses of variance by ANOVA if Bartlett’s test of heterogenicity indicated homo- geneity of variances and by the Kruskal–Wallis test if Bartlett’s test was significant at the 5% level. Results Analysis of baseline data Of 1137 children examined at the baseline, 904 (79.5%) returned stool samples at follow-up. Loss at follow-up was high compared with that in other surveys in Pembian schoolchildren because of an unexpected closure of schools on Pemba Island one week before the scheduled 21-day follow-up. The schools were closed for three weeks to allow children to help harvest an unusually large crop of cloves. The research staff thus walked from hut to hut through each village to trace missing children. Subsequent analysis showed that older, male children were more likely to be lost to follow-up, presumably because of their increased involvement in clove picking (12.2 vs 11.6 years, P<0.001; 54.5% boys vs 47.0% girls, P<0.05) (Table 2). Children subsequently lost at follow- up had similar baseline prevalence and intensity of A. lum- bricoides, T. trichiura, and hookworm infections to children who were followed up. Results are presented on the cohort of 904 children who returned a stool sample for the baseline and post-treatment surveys (Table 3). Their mean (SD; range) age was 11.5 (2.4; 7– 18) years, and 45% were boys. The four groups were homogeneous at baseline for age and helminth infections, but differed in the proportion of males to females (Table 2). The prevalence of any helminth infection was 99.7%, with 38.3% and 54.9% of children harbouring double and triple infections, respectively. Table 3 gives the prevalence and intensity of A. lumbricoides, T. trichiura, and hookworms at baseline and 21 days after treatment in the four treatment groups. Overall drug efficacy Follow-up egg counts, cure rates, reductions in prevalence and egg reduction rates for the three nematode infections were statistically significantly better with all of the drug regimens compared with those at baseline, except for cure rates for hookworm infections with mebendazole and for T. trichiura infections with levamisole (although in both cases the mean egg counts were reduced substantially). Compared with placebo, all drug treatments produced significantly higher cure rates and egg reduction rates and lower prevalences at follow- up, except for the egg reduction rate for levamisole in T. trichiura infections (Table 3). Both drugs had very high efficacy (98.5% and 99.1% egg reduction rates for levamisole and mebendazole, respectively) against A. lumbricoides. Mebendazole alone and in combination with levamisole had better efficacy than levamisole alone for T. trichiura infection (81% and 85% vs 41.5% egg reduction rates, P<0.001). Levamisole treatment produced a marginally significant reduction in prevalence of hookworm infection, which was greater than the reduction seen with mebendazole (8.9% vs 3.6%, P<0.05); the combination had better efficacy in reducing prevalence than either drug alone (23.6%, P<0.001). The egg reduction rate for hookworm infection was 88.7% for the combined treatment, but significantly less for either drug alone (61.3% for levamisole and 52.1% for mebendazole, P<0.001). Figure 1 shows the distribution of intensities of infections, expressed as eggs per gram faeces, before and after treatment for A. lumbricoides, T. trichiura, and hookworm infections. Higher efficacy of treatment is indicated by an increase in negative and light infections and a decrease in moderate and heavy infections 21 days after treatment. Stratified analysis by intensity was performed on the cure rates and egg reduction rates for each species of helminth. For any treatment, lower cure rates were achieved in children with heavy infections compared with light infections:A. lumbricoides 93.3% vs 97.0% (P=0.07), T. trichiura 8.3% vs 19.3% (P=0.1), and hookworm 8.4% vs 16.3% (P<0.05). On the other hand, higher egg reduction rates were achieved in childrenwith heavy compared with light infections for T. trichiura (93.8% vs 81.4%, P<0.05) and hookworm (91.3% vs 73.2%, P<0.01). Efficacy of treatment was not influenced by whether A. lumbricoides, T. trichiura, or hookworm infections presented as single or multiple infections. Although adverse effects were not investigated actively, no adverse events were reported after any single or combined treatment in the week following the administration of anthelminthics. Drug efficacy in Standard 1 and Standard 5 children Overall, 446 childrenwere in Standard 1 classes and had amean (SD) age of 9.5 (1.5) years, and 458 children were in Standard Table 1. Egg counts (eggs per gram) used to describe intensity of infection Causative pathogen Intensity of infection (egg count per gram) Light Moderate Heavy A. lumbricoides 1–4999 5000–9999 510 000 T. trichiura 1–999 1000–9999 510 000 Hookworm 1–1999 2000–3999 5 4000 345Bulletin of the World Health Organization 2003, 81 (5) Mebendazole and levamisole alone or in combination for nematode infection in Zanzibar 5 classes and had a mean (SD) age of 13.4 (1.3) years. Boys accounted for 49.8% and 40.4% of children from Standard 1 and Standard 5 classes, respectively. Tables 4 and 5 give the prevalences and intensities of A. lumbricoides, T. trichiura, and hookworm infections at baseline and 21 days after treatment in the four treatment groups in Standard 1 and Standard 5 children, respectively. The baseline mean intensities of infection for all three species of helminths were significantly higher in the Standard 1 children than in the Standard 5 children (P<0.001). In addition, Standard 1 children at baseline had higher prevalences of moderate and heavy infection for each helminth than Standard 5 children — (A. lumbricoides: moderate 14.1% vs 9.2%, heavy 18.8% vs 9.0%; T. trichiura: moderate 45.3% vs 22.3%, heavy 2.5% vs 0.2%; and hookworm: moderate 12.1% vs 6.3%, heavy 11.4% vs 5.0%). In both Standard 1 and Standard 5 children, but particularly in Standard 1 children, mebendazole and its combination with levamisole were more effective than levamisole in T. trichiura infection (the efficacy of levamisole did not differ significantly from that of placebo). In Standard 1 Table 2. Characteristics of the study sample at baseline Characteristic Treatment Mebendazole Levamisole Mebendazole + levamisole Placebo Total Lost at follow-up Total Lost at follow-up Total Lost at follow-up Total Lost at follow-up (n = 285) (n = 49) (n = 277) (n = 67) (n = 286) (n = 70) (n = 289) (n = 47) Mean age (years ) 11.5 (2.3)a 12.3 (2.1) 11.7 (2.5) 12.1(2.3) 11.7 (2.4) 12.2 (2.5) 11.6 (2.5) 12.1 (2.3) Sex (% boys) 48.4 53.1 54.2 56.7 43.0 55.7 42.6 51.1 Ascaris Prevalence (%) 61.4 69.4 60.3 62.7 62.9 65.7 58.1 63.8 Geometric mean (epg)b 134 292 99 117 129 149 100 122 Trichuris Prevalence (%) 90.9 91.8 94.6 97.0 93.7 95.7 93.8 93.6 Geometric mean (epg) 304 315 365 384 371 421 458 346 Hookworm Prevalence (%) 95.1 95.9 96.0 95.5 95.1 98.6 95.2 89.4 Geometric mean (epg) 429 352 454 392 401 504 475 316 a Values in parentheses are standard deviations. b epg = eggs per gram. Table 3. Results before and 21 days after treatment with mebendazole, levamisole, mebendazole + levamisole, and placebo Causative Prevalencea Egg count (epg)b pathogen Drug n Day 0 Day 21 Day 0 Day 21 Cure Reduction P-valuec Egg reduction P-valuec rate in prevalence rate Ascaris Mebendazole 236 59.7 3.0 114 0.2 96.5 95.0 <0.001 99.0 (98.2–99.4)d <0.001 Levamisole 210 59.5 5.7 94 0.4 91.2 90.4e <0.001 98.5 (97.4–99.1) <0.001 Mebendazole + levamisole 216 62.0 1.4 131 0.1 98.5 97.7 <0.001 99.1 (98.4–99.5) <0.001 Placebo 242 57.0 51.2 96 63 22.5 10.2 NSf 33.9 (0.4–56.1) NS Total 904 59.5 106 Trichuris Mebendazole 236 90.7 75.0 302 57 22.9 17.3g <0.001 81.0 (71.9–87.1)g <0.001 Levamisole 210 93.8 90.0 359 210 9.6 4.1h NS 41.5 (17.8–58.4)h <0.01 Mebendazole + levamisole 216 93.1 74.5 356 53 22.9 20.0 <0.001 85.0 (78.5–89.5) <0.001 Placebo 242 93.8 94.2 484 395 4.8 –0.4 NS 18.3 (–7.0–37.6 ) NS Total 904 92.8 371 Hookworms Mebendazole 236 94.9 91.5 447 213 7.6 3.6h,i NS 52.1 (36.0–64.2)h <0.001 Levamisole 210 96.2 87.6 476 184 11.9 8.9h <0.001 61.3 (47.2–71.6)h <0.001 Mebendazole + levamisole 216 94.0 71.8 373 41 26.1 23.6 <0.001 88.7 (83.9–92.0) <0.001 Placebo 242 96.3 95.9 514 432 3.4 0.4 NS 16.0 (–8.0–34.6) NS Total 904 95.4 451 a Percentage positive for pathogen. b Mean egg count expressed as geometric mean. c P-value from day 21 to day 0. d Values in parentheses are 95% confidence intervals. e Compared with mebendazole plus levamisole P<0.05. f NS, not significant. g Compared with levamisole P<0.001. h Compared with mebendazole plus levamisole P<0.001. i Compared with levamisole P<0.05. 346 Bulletin of the World Health Organization 2003, 81 (5) Research 347Bulletin of the World Health Organization 2003, 81 (5) Mebendazole and levamisole alone or in combination for nematode infection in Zanzibar children, the effect of mebendazole against hookworm infection was similar to that of placebo, although its reduction of intensity of infection approached statistical significance (egg reduction rate 37%, P = 0.06), while levamisole was more effective (egg reduction rate 66.3%, P<0.001) and the combination was the best treatment (egg reduction rate 92.9%, P<0.001). In Standard 5 children, the combination was still the better option (although to a lesser extent than in Standard 1 children) than either drug alone for the treatment of hookworm infection (egg reduction rate 81.9% vs 56.3% for levamisole and 60.4% for mebendazole, P<0.05). Overall, none of cure rate, percentage reduction in prevalence, or egg reduction rate differed significantly between Standard 1 and Standard 5 children for any helminth infection. Furthermore, no evidence was seen of a reduced cure or egg reduction rate for mebendazole relative to levamisole in Standard 5 children compared with Standard 1 children. Discussion This study confirms the extremely high prevalence of intestinal nematode infections in Zanzibar despite the periodic che- motherapy control programme that started in 1994. The objective of this programme, however, was reduction in intensity rather than prevalence of infection. In this respect, the significantly lower egg counts at baseline in Standard 5 children who had received 15 rounds of treatment with mebendazole compared with children in Standard 1 classes, who had never been treated, are encouraging. A possible explanation for this finding could be an intrinsic decline in intensity with increasing age. This is not supported by a previous study on untreated children in the same population, however, which showed that between the ages of 9 (Standard 1) and 13 years (Standard 5), the mean intensity of ascariasis in fact increased, the intensity of hookworm infections remained stable, and the intensity of trichuriasis slightly decreased, although to a much lesser extent than between Standard 1 and Standard 5 children in the present study (21). The benefit of periodic chemotherapy is shown further in the significant reduction of moderate and heavy helminth infections — those most associated with morbidity — in Standard 5 children. From the public health perspective, it seems, therefore, that the helminth control programme is effective in reducing intensity, although the low efficacy of treatment of hookworm infections found in this trial is worrying. High, and similar, levels of efficacy in the treatment of A. lumbricoides infection were achieved using mebendazole, levamisole, and their combination. The efficacy of each treatment was equally excellent in light and heavyA. lumbricoides infections, with egg reduction rates approaching 100%. This result is consistent with previous efficacy trials and is also similar — for mebendazole — to the results of an efficacy study carried out in Pemba in 1993 before the mebendazole- based control programme was started (14, 24–27). Drug efficacies inT. trichiura infectionwere also similar to those seen in previous trials, as treatment with mebendazole reduced the prevalence of infection, while levamisole had a much smaller effect (14, 26–29). Reductions in egg counts were significant for mebendazole and for levamisole, although mebendazole and the combined treatment were both significantly better than levamisole alone. When compared with the efficacy trial performed in Pemba before the control programme started, the mebendazole cure and egg reduction Table 4. Results before and 21 days after treatment with mebendazole, levamisole, mebendazole + levamisole, and placebo in Standard 1 children Causative Prevalencea Egg count (epg)b pathogen Drug n Day 0 Day 21 Day 0 Day 21 Cure Reduction P-valuec Egg reduction P-valuec rate in prevalence rate Ascaris Mebendazole 120 67.5 5.8 264 0.4 93.8 91.4d <0.001 99.5 (98.8–99.7)e <0.001 Levamisole 98 70.4 4.1 254 0.4 94.2 94.2f <0.001 99.5 (98.8–99.8) <0.001 Mebendazole + levamisole 108 67.6 0.1 248 0.1 100.0 99.9 <0.001 99.6 (99.1–99.8) <0.001 Placebo 120 65.0 61.7 192 152 17.9 5.1 NSg 20.9 (–44.1–56.6) NS Total 446 67.5 237 Trichuris Mebendazole 120 95.0 76.7 534 69 22.8 19.3h <0.001 86.9 (78.0–92.2)h <0.001 Levamisole 98 95.9 95.9 643 416 4.2 0d NS 35.2 (–3.7–59.5)d NS Mebendazole + levamisole 108 97.0 82.4 776 94 17.1 15.1 <0.001 87.9 (80.6–92.4) <0.001 Placebo 120 95.0 95.8 726 590 4.4 –0.8 NS 18.8 (–18.8–44.5) NS Total 446 95.7 661 Hookworms Mebendazole 120 94.1 95.0 483 304 4.4 –1.0i NS 37.0 (6.0–57.8)d,i 0.06 Levamisole 98 99.0 90.8 827 278 9.2 8.3d <0.01 66.3 (48.7–7.8)d <0.001 Mebendazole + levamisole 108 96.3 71.3 604 42 26.0 26.0 <0.001 92.9 (88.7–95.6) <0.001 Placebo 120 96.7 98.3 674 619 0.9 –1.7 NS 8.1 (–29.0–34.6) NS Total 446 96.4 628.0 a Percentage positive for pathogen. b Mean egg count expressed as geometric mean. c P-value from day 21 to day 0. d Compared with mebendazole plus levamisole P<0.01. e Values in parentheses are 95% confidence intervals. f Compared with mebendazole plus levamisole P<0.05. g NS, not significant. h Compared with levamisole P<0.001. i Compared with levamisole P<0.05. 348 Bulletin of the World Health Organization 2003, 81 (5) Research rates were not significantly different (24). The poor efficacy of levamisole against T. trichiura has already been reported, although Ismail et al. showed an egg reduction rate of 73% (2, 14). The finding that egg reduction rates were significantly higher in both T. trichiura and hookworm ‘‘heavy’’ infections compared with ‘‘light’’ infections is consistent with trials with albendazole (29, 30). Striking features of this study were the low cure (7.6%) and egg reduction (52.1%) rates when mebendazole was used against hookworms. Mebendazole efficacy was lower when compared with that in published studies and, more specifically, compared with that in the trial carried out in Pemba before exposure to periodic treatment, in which mebendazole had a cure rate of 22.4% and an egg reduction rate of 82.4% (14, 24– 27). Furthermore, in a recent trial comparable with our trial in the neighbouring island of Mafia, in which there is no school- based deworming programme and use of benzimidazoles has been very limited, mebendazole efficacies were similar (cure rate 31.3%; egg reduction rate 78.1%) to the pre-treatment values on Pemba and very much higher than the current efficacy in Pemba (31). A number of potential confounding factors were ruled out by the fact that both trials were carried out in school- children by the same staff and used the samemethods and drug (mebendazole 500mg from Janssen). It is notable that the cure rates and egg reduction rates for Ascaris and Trichuris were comparable between the two trials. Interestingly, levamisole also showed a lower efficacy against hookworms in the present study than in previous studies (32–34). A possible explanation is that levamisole has been reported to be more effective againstA. duodenale thanN. americanus, but the latter pathogen is the most prevalent species in Pemba (20). If mebendazole also was less effective against N. americanus, repeated use of mebendazole in Pemba could have created an increased Necator:Ancylostoma ratio, which would have resulted in an overall lower efficacy against hookworms. Data from recent studies suggest that the Necator:Ancylostoma ratio is indeed higher now than it was before periodic anthelminthic treatment (Albonico, unpublished data, 2000) (20). An alternative explanation for the apparent reduced efficacy of mebendazole compared with historical controls is the selection of drug resistance by the repeated treatment regimen. If a proportion of drug-resistant worms was present in the worm population in Pemba, we would have expected resistant worms to have accumulated in children after multiple rounds of treatment and reinfection and to have represented a higher proportion of worms than that found in Standard 1 children. In this case, a lower drug efficacy would have been expected in the Standard 5 children. In fact, the efficacy of mebendazole in Standard 5 children who had receivedmultiple (15) doses of mebendazole was not lower than in Standard 1 children who had never been treated; this suggests that a mebendazole-resistant population of worms had not accumu- lated in the repeatedly treated Standard 5 children. Never- theless, the possibility that drug resistance is emerging in Pemba cannot be excluded. In Mali, failure of mebendazole in the treatment of human hookworm infections has been reported (35). In that case, however, the population had not been exposed to periodic treatment with mebendazole, although mebendazole had been available in that community for some years; pre-exposure efficacy data were not available for comparison and the sample of children studied was rather small. These factors suggest a need for caution when interpreting these results. A subsequent drug efficacy study in Mali (36) showed reasonably good efficacy of mebendazole and even better results from albendazole, which show a lack of significant resistance in human hookworms to benzimidazoles in that area. Poor efficacy of pyrantel against hookworms has Table 5. Results before and 21 days after treatment with mebendazole, levamisole, mebendazole + levamisole, and placebo in Standard 5 children Causative Prevalencea Egg count (epg)b pathogen Drug n Day 0 Day 21 Day 0 Day 21 Cure Reduction P-valuec Egg reduction P-valuec rate in prevalence rate Ascaris Mebendazole 116 51.7 0.1 47 0.1 100.0 99.8d <0.001 97.9 (95.8–99.0) <0.0001 Levamisole 112 50.9 7.1 39 0.5 87.5 86.1e <0.001 96.1 (92.0–98.1) <0.0001 Mebendazole + levamisole 108 56.5 2.8 61 0.2 96.7 95.0 <0.001 98.0 (95.7–99.1) <0.0001 Placebo 122 49.2 41.0 48 26 28.3 16.7 NS 44.5 (3.8–68.0) NS Total 458 51.7 48 Trichuris Mebendazole 116 86.2 73.3 167 46 23.0 15.0 <0.01 72.1 (51.1–84.1) <0.001 Levamisole 112 92.0 84.8 215 115 14.6 7.8f NS 46.5 (12.6–67.2)e <0.05 Mebendazole + levamisole 108 88.9 66.6 163 30 29.2 25.1 <0.001 81.4 (68.0–89.1) <0.001 Placebo 122 92.6 92.6 325 266 5.3 0.0 NS 17.8 (–19.0–43.2) NS Total 458 90.0 211 Hookworms Mebendazole 116 95.7 87.9 412 148 10.8 8.2e <0.05 60.4 (45.2–76.4)e <0.001 Levamisole 112 93.7 84.8 293 128 14.3 9.5e <0.05 56.3 (30.7–72.4)e <0.01 Mebendazole + levamisole 108 91.7 72.2 230 41 25.3 21.3 <0.001 81.9 (70.1–89.0) <0.001 Placebo 122 95.9 93.4 395 304 6.0 2.6 NS 23.0 (–11.5–46.8) NS Total 458 94.3 327 a Percentage positive for pathogen. b Mean egg count expressed as geometric mean. c P-value from day 21 to day 0. d Compared with levamisole P<0.001. e Compared with mebendazole plus levamisole P<0.05. f Compared with mebendazole plus levamisole P<0.001. 349Bulletin of the World Health Organization 2003, 81 (5) Mebendazole and levamisole alone or in combination for nematode infection in Zanzibar been found in north-west Australia, but again it is not known if this represents a decline in efficacy because of emerging resistance (37). The efficacy of the combined administration of mebendazole 500 mg and levamisole 40 or 80 mg was evaluated for the first time in this study and showed higher efficacy than either drug alone against hookworm infections. This is a promising result, because the combined administra- tion of two different anthelminthic drugs could be used as the treatment of choice in this context. In addition, the adoption of combination therapy with drugs with distinct modes of action when used at early stages has been shown to delay the onset of anthelminthic drug resistance (18). Although this study clearly shows that the efficacy of mebendazole in the treatment of hookworm infections in Pemba Island schoolchildren is lower than in previous studies, demonstration of anthelminthic resistance is still lacking. A need exists, therefore, for alternative methods by which to address this question, such as the in vitro egg hatch assay — a technique widely used in veterinary medicine (38) and recently adapted to human hookworms (39). This would require comparisons with strains of hookworms that had not been exposed to treatment. The early detection of drug resistance is of the utmost importance, because the in vivo and in vitro tests currently available in the veterinary field detect resistance when the proportion of worms carrying the drug resistance allele in a population already has reached >25% (40). The development of methods for the early detection of benzimidazole resistance in human nematodes would be of great value, therefore, and the polymerase chain reaction methods developed by veterinary scientists need to be evaluated for their applicability in human nematodes (41). n Acknowledgements Special thanks to Professor Peter Smith for his valuable advice on statistical analysis, and for the revision of the manuscript. The authors thank also Dr Nerissa Kohen and Victoria Wright for their contribution towards the data collection in Pemba Island. We are grateful to the staff of the Helminth Control Programme, as well as the staff of the PublicHealth Laboratory Ivo de Carneri in Pemba Island, whose dedication and enthusiasm made this study possible. We acknowledge the donation of placebo and mebendazole from Pharmamed (Malta) and Janssen (Belgium) and of levamisole from Zeneca (UK). This study was supported generously by Parasitic Diseases and Vector Control, Division of Communicable Diseases Control, Prevention and Eradication, World Health Organization, Geneva. Conflicts of interest: none declared. Re´sume´ Efficacite´ du me´bendazole et du le´vamisole seuls ou en association contre les ne´matodoses intestinales apre`s traitement pe´riodique cible´ par le me´bendazole a` Zanzibar Objectif Evaluer l’efficacite´ du me´bendazole (500 mg) et du le´vamisole (40 ou 80 mg) et la re´sistance a` ces compose´s administre´s seuls ou en association pour le traitement des infections a` Ascaris lumbricoides, Trichuris trichiura et les ankylostomes sur l’ıˆle de Pemba, une re´gion ou` sont effectue´s depuis 1994 des traitements pe´riodiques par le me´bendazole en milieu scolaire. Me´thodes Un essai controˆle´ randomise´ contre placebo a e´te´ re´alise´ sur 914 enfants des premier et cinquie`me niveaux de l’enseignement primaire. Des e´chantillons de selles recueillis au de´but de l’e´tude et 21 jours apre`s le traitement ont e´te´ examine´s par la technique de Kato-Katz afin d’e´valuer la pre´valence et l’intensite´ des helminthiases. Re´sultats L’efficacite´ du me´bendazole et du le´vamisole en traitement unique des ne´matodoses intestinales e´tait comparable a` celle observe´e lors de pre´ce´dents essais, mais le traitement des ankylostomiases par le me´bendazole donnait des taux de gue´rison (7,6 %) et de re´duction du nombre d’œufs (52,1 %) significative- ment plus faibles que lors d’une e´tude re´alise´e avant le de´but de la chimiothe´rapie pe´riodique (taux de gue´rison : 22,4 % ; taux de re´duction du nombre d’œufs : 82,4 %). Le traitement associe´ par le me´bendazole et le le´vamisole e´tait significativement plus efficace contre les ankylostomiases (taux de gue´rison : 26,1 % ; taux de re´duction du nombre d’œufs : 88,7 %) qu’un traitement par chacun des me´dicaments administre´s se´pare´ment. Aucune diffe´rence d’efficacite´ du me´bendazole n’a e´te´ observe´e entre les enfants ayant rec¸u une chimiothe´rapie pe´riodique et ceux qui n’avaient jamais e´te´ traite´s auparavant. Conclusion Il semble d’apre`s les re´sultats que l’efficacite´ globale du me´bendazole contre les ankylostomiases apre`s chimiothe´rapie pe´riodique soit diminue´e. L’efficacite´ des benzimidazole´s dans le cadre des programmes de lutte reposant sur la chimiothe´rapie doit eˆtre e´troitement surveille´e. Un traitement associant le me´benda- zole et le le´vamisole peut eˆtre utile pour retarder le de´veloppement de la re´sistance a` cette cate´gorie de me´dicaments. Resumen Eficacia del mebendazol y el levamisol, solos o combinados, contra las infecciones intestinales por nematodos despue´s del tratamiento repetido focalizado con mebendazol en Zanzı´bar Objetivo Evaluar la eficacia del mebendazol (500 mg) y el levamisol (40 u 80 mg), solos o combinados, y la resistencia a ellos como tratamiento de las infecciones por Ascaris lumbricoides y Trichuris trichiura y de la anquilostomiasis en la Isla de Pemba, una zona en la que desde 1994 se llevan a cabo perio´dicamente tratamientos con mebendazol en las escuelas. Me´todos Se llevo´ a cabo un ensayo aleatorizado y controlado mediante placebo en 914 nin˜os inscritos en alguno de los cinco primeros an˜os de escuela primaria. Las muestras de heces obtenidas en la situacio´n de partida y a los 21 dı´as de tratamiento fueron analizadas mediante la te´cnica de Kato-Katz a fin de evaluar la prevalencia y la intensidad de las infecciones por helmintos. Resultados La eficacia del mebendazol y el levamisol como tratamientos u´nicos de las infecciones intestinales por nematodos fue comparable a la observada en ensayos anteriores, pero la administracio´n de mebendazol contra las 350 Bulletin of the World Health Organization 2003, 81 (5) Research infecciones por anquilostoma logro´ unas tasas de curacio´n (7,6%) y de reduccio´n del nu´mero de huevos (52,1%) significativamente inferiores a las notificadas en un estudio emprendido antes del comienzo de la antibioticoterapia perio´dica (tasa de curacio´n, 22,4%; tasa de reduccio´n del nu´mero de huevos, 82,4%). El tratamiento combinado con mebendazol y levamisol tuvo una eficacia significativamente mayor contra las infecciones por anquilostoma (tasa de curacio´n, 26,1%; tasa de reduccio´n del nu´mero de huevos, 88,7%) que cualquiera de los medicamentos por separado. 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