Epidemiological evidence for a differential effect of hookworm species, Ancylostoma duodenale or Necator americanus, on iron status of children

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1 C International Epidemlologial Assoiation 1998 Printed in Great Britain International Journal of Epidemiology 1998,27: Epidemiologial evidene for a differential effet of hookworm speies, Anylostoma duodenale or Neator amerianus, on iron status of hildren M Albonio, a RJ Stoltzfus, b L Savioli, JM Tielsh, d HM Chwaya, e E Erole f and G CanriniS Bakground The hookworms, Anylostoma duodenak and Neator amerianus, ause signifiant gastrointestinal blood loss. In linial studies, greater blood losses have been reported with A. duodenak. However, there has been no evidene that endemi A. duodenak infetion has greater impat than N. amerianus infetion on the iron status of populations. Methods In a sample of 525 shool hildren in Pemba Island, Tanzania, we ompared the degree of anaemia and iron defiieny assoiated with the two hookworm speies at the individual and ommunity (i.e. shool) levels. Multiple regression was used to ontrol for infetion intensities and other hild harateristis. Results In the 492 hildren with hookworm positive faeal ultures, haemoglobin and ferritin onentrations dereased with inreasing proportions of A. duodenak. Among hildren with only N. amerianus larvae, the prevalene of anaemia was 60.5% and the prevalene of ferritin <12 (Xg/1 was 33.1%, while in hildren with 3=50% A. duodenak larvae, the respetive prevalenes were 80.6% and 58.9%. When hildren were grouped by the prevalene of A. duodenak at the shool level, hildren from high prevalene (3=20%) shools had signfiantly worse iron defiieny and anaemia than hildren from low prevalene shools. Conlusions The speies of hookworm being transmitted in a ommunity influenes the burden of iron defiieny anaemia in the ommunity, and should be onsidered in prioritizing and planning programmes for hookworm and anaemia ontrol. Keywords Hookworms, Anylostoma duodenak, Neator amerianus, shool hildren, iron defiieny, anaemia Aepted 7 Otober 1997 Hookworms infet an estimated 1.3 billion people globally. 1 Iron-defiieny anaemia aused or exaerbated by intestinal blood loss is the most outstanding feature of hookworm infetion. Blood loss is aused when hookworms attah to a Division of Communiable Diseases. World Health Organization, Geneva. Switzerland. b Center for Human Nutrition, Shool of Hygiene and Publi Health, The Johns Hopkins University, 615 N Wolle St. Baltimore, MD 21205, USA. Division of Control of Tropial Diseases, World Health Organization, Geneva, Switzerland. d Department of International Health, The Johns Hopkins Shool of Publi Health, Baltimore, USA. r Ministry of Health, Zanzibar, United Republi of Tanzania 1 Pemba Helminth Control Programme, Zanzibar, United Republi of Tanzania. g Department of Parasitology, University 'La Sapienza', Rome, Italy Reprint requests to Rebea J Stoltzfus. and feed from the intestinal muosa, and the magnitude of blood loss is diretly related to the number of adult worms residing in the intestine. 4 The balane between iron intake and iron loss from intestinal bleeding determines the risk and severity of iron-defiieny anaemia from hookworm infetion. 5 There are primarily two speies of hookworms that infet humans; Anylostoma duodenak and Neator amerianus. 6 Neator amerianus tends to thrive in more tropial limates, while A. duodenak favours ooler, drier limates. However, their geographial distributions overlap onsiderably, and both speies are endemi to many areas. 2 Hookworms are transmitted when hookworm eggs exreted in human faees are inubated in appropriate soil onditions, hath into larvae and enter a human host through the skin. In ontrast to N amerianus, A duodenak an infet humans by the oral as well as the perutaneous route 7 and may undergo arrested development in human organ systems in its larval stage

2 HOOKWORM SPECIES AND IRON STATUS 531 From a morbidity standpoint, the most important differene between the two speies is that A. duodenale auses approximately five times greater blood loss than N. amerianus. 2 This has been demonstrated in linial studies in whih isotopially labelled red blood ells were injeted into hookworm-infeted individuals, and the faeal exretion of the label was measured before and after deworming. Estimates from individual studies have found 2- to 10-fold higher blood losses with A. duodenale than with N. amerianus. 4 However, there has not been evidene that endami A. duodenale infetion has greater impat than N. amerianus infetion on the iron status of populations. The population biology of hookworms, the host-parasite interation, 10 and iron absorption in humans 11 may adapt in ways that prevent the burden of hookworm anaemia from beoming overly great in stable human populations. 12 Suh ompensatory strategies may prevent speies related differenes in blood loss per hookworm from manifesting into differenes in the prevalene and intensity of iron-defiieny anaemia in human populations. Beause of the diffiulty of measuring atual numbers of worms in population studies, most studies of hookworm infetion and iron-defiieny anaemia in ommunities have used faeal egg ounts to determine the presene and severity of hookworm infetion. By this method, the two hookworm speies annot be distinguished, beause their eggs have similar mirosopi appearane. 2 Thus, there has been no epidemiologiaj evidene that the speies of hookworm being transmitted in a ommunity has a meaningful impart on the iron status of the population. We have studied the epidemiology of hookworm infetion and iron-defiieny anaemia in shoolhildren in Pemba Island, Zanzibar. 13 Hookworms are highly endemi on the island, 14 and both speies were believed to be present. 15 In the ourse of an ongoing evaluation of the impart of shool-based deworming on hildren's nutritional status, we determined the speies of hookworm infetion in a sample of 525 hildren from 10 randomly seleted ommunities. Infetion intensities and iron status were also assessed. Our objetives were: (1) to desribe the distribution of the two hookworm speies aross the island, (2) to determine whether hildren infeted with A. duodenale have worse iron status than those infeted with N. amerianus, and (3) to demonstrate whether ommunities where A. duodenale is highly endemi have greater burdens of iron-defiieny anaemia than ommunities primarily infeted with N. amerianus, ontrolling for other fators. Methods Study area and sample Pemba Island, the smaller of the two islands of Zanzibar, United Republi of Tanzania, lies 40 km off the East Afrian oast, 5 S of the equator and is lassed as a ontinental island with humid tropial limate. The northeast monsoon blows from Otober to Deember and is haraterized by short rains. The southwest monsoon brings the long rains from mid-marh to mid-may. The temperatures rarely exeed 34 or fall below 21 C. Pemba is densely populated with 330 inhabitants per km 2. The island is divided into four distrits of roughly equal size. About people live in eah of the three main towns of Wete, Chake-Chake and Mkoani, the rest of the population being sattered in numerous villages. The eonomy of the inhabitants is mainly based on farming and fishing. The primary rops onsist of subsistene agriulture, with some love and oonut plantations. The sample for the present study was seleted from hildren inluded in the baseline evaluation survey for the Zanzibar shool-based deworming programme. For the evaluation, 12 shools were randomly seleted stratified by distrit, and 3605 hildren in morning lasses of grades 1 4 in those shools partiipated in the survey. 13 The sample for the present study inluded 570 hildren, drawn as a systemati random subsample. Speifially, every seventh hild surveyed from 10 of the 12 shools was inluded. Children from the first shool were not inluded beause the method for ulturing hookworm larvae was not yet in plae, and hildren from the last shool were not inluded beause we had already exeeded our intended sample size of 500 hildren. The study was approved by the internal review boards of The Johns Hopkins University, the Ministry of Health of Zanzibar, and the World Health Organization. Assessments Survey data were olleted in the shools by speially trained staff of the Ministry of Health of Zanzibar. On the day before the survey, plasti ontainers were distributed to eah lass and the hildren were asked to bring a stool sample to shool the next day. Over 95% of hildren returned a faeal sample. Faeal hookworm egg ounts were determined by the Kato-Katz tehnique. 16 To determine hookworm speies, ultures were suessfully obtained for 525 of the 570 hildren seleted for the study. The Harada-Mori tehnique 17 modified aording to Garia & Brukner 18 was used in order to inrease the effiieny of the ulture method. Approximately 1 g faees was mixed with ative granular haroal, and some water was added in the ase of hard stools, to obtain an homogeneous paste. Two mirosope slides were folded in a piee of absorbent paper and plaed in a Petri dish with a diameter of 9 m. The stool speimens were smeared arefully on the surfae of the absorbent paper and 5-6 ml of distilled water were added to the Petri dish, without reahing the stool level. The ultures were overed and labelled and left at room temperature (26-30 C) in a shady plae. The positivity of the ultures was heked with a disserting mirosope and after 7-10 days the ultures were transferred to a test tube. The larvae were killed by heating (100 C) in water for 2-3 min and then fixed with 10% formalin. The sediment was measured and a representative sample (usually 1/3 or 1/4) was examined for larvae that were identified and ounted. The ounts were then multiplied by 3 or 4 aording to the proportion of sediment examined. Urine samples were olleted from 100% of hildren, and mirohaematuria was diagnosed using Hemastix test strips (Ames Laboratories, In., Elkhart, IN). In this population mirohaematuria sreens for urinary shistosomiasis (Shistosoma haematobium infetion) with 69% sensitivity and 89% speifiity. 19 Blood samples were olleted by venipunture from all hildren to assess iron status. Haemoglobin was determined by the HemoCue method (HemoCue AB, Angelhom, Sweden) and erythroyte protoporphyrin was determined using a front-fae haematofluorometer (Aviv Biomedial In., Lakewood, NJ). A thin blood film was stained with Giemsa and malaria parasites

3 532 INTERNATIONAL JOURNAL OF EPIDEMIOLOGY were quantitated against leukoytes using standard methods. The remaining blood was allowed to lot at room temperature, and serum was olleted by entrifugation. Sera were stored at 10 C for up to 8 weeks, transported to Baltimore in liquid nitrogen and stored at -70 C for up to 4 months. Serum ferritin was determined on 442 samples (84% of study hildren) using a fluoresene-linked immunoassay (DELFIA System by Walla, In., Gaithersburg, MD). Missing data for serum ferritin were mainly due to mislabelling of speimens, and losses in transport. rs: 80 o 60 ra 40 i a- 20 Statistial analysis 1 o.ill E N. amerianus A. duodenale 3 o f C ra w Shool Figure 1 Prevalene ol N. amerianus and A duodenale infetions in hildren from 10 shools on Pemba Island, Tanzania. Number of hildren surveyed in eah shool ranges from 35 to 60 orret the faeal egg ounts from the Kato-Katz method to reflet the relative worm burdens of the two speies.2 Beause a given number of eggs/g faees represents fewer A. duodenale worms than N. amerianus worms, ontrolling for hookworm eggs/g faees might bias against finding a true speies-related differene in iron status. However, it protets us from attributing to speies a differene in iron status that was truly aused by greater worm burdens. We arried out analyses with and without adjustment for hookworm eggs/g faees, and found it hanged the results very little. We present results of analyses with adjustment for hookworm eggs/g faees, a onservative approah. To adjust for hookworm eggs/g faees, an ordinal ategorial variable was inluded in the multivariate regression models, oded as 0 if 0 eggs/g, 1 if eggs/g, 2 if eggs/g, and 3 if eggs/g, and 4 if s*6000 eggs/g faees. The multiple R2 for the models were highest when we used the variable in this form, rather than geometri mean eggs/g or other ategorial forms. Data were analysed using Systat statistial software (SPSS, In., Chiago, IL). Results Prevalene and geographial distribution of hookworm speies Based on the faeal ultures, 33 hildren (6.3%) were not infeted with hookworms, 11 (2.1%) were infeted with only A. duodenale, 340 (64.8%) were infeted with only N. amerianus, and 141 (26.9%) were infeted with both speies of hookworm (Table 1). The prevalene of hookworm infetion based on faeal ultures was 93.7%, and based on Kato-Katz method was 96.2%. The overall agreement for hookworm positivity between the faeal ulture and Kato-Katz methods was 91.4% (480/525). Only four of the 525 hildren studied, less than 1% of the sample, were hookworm negative by both methods. Distributions of faeal egg ounts, serum ferritin and erythroyte protoporphyrin were skewed to high values, so geometri means are presented. Anaemia was defined as haemoglobin <11 g/dl. This is lower than the WHO-reommended utoff for anaemia, but a lower utoff sreens more effiiently for iron-defiieny anaemia in blak populations. A haemoglobin utoff of 9 g/dl was used to define moderate-severe anaemia. Cutoffs for protoporphyrin are not well-defined. In these analyses values >120 (imol/mol heme were onsidered indiative of severe iron-defiient erythropoiesis. Serum ferritin <12 mg/1 was used to indiate exhausted iron stores. The prevalenes of A. duodenale and N. amerianus infetions were examined for the entire sample, and by shool. To ompare the effet of the two speies in individual hildren, hildren with hookworm positive larval ultures were grouped into those with only N. amerianus larvae, those with 1-50% A. duodenale larvae, and those with >50% A. duodenale larvae. The latter group inludes 11 hildren with apparently pure A. duodenale infetions (i.e. no N amerianus larvae were found). Neator amerianus was highly prevalent in all ommunities surveyed, but the prevalene of A. duodenale ranged from 2% to 88% (Figure 1). To examine the effet of speies on ommunity burden of iron-defiieny anaemia, hildren in four shools with >20% prevalene of A. duodenale were ompared to hildren in six shools with <20% prevalene. In both individual and ommunity level analyses, multiple regression was used to test the effet of hookworm speies on measures of iron status. In individual level analyses the hookworm speies variable was an ordinal variable with three values: 0 if pure N. amerianus, 1 if 1-50% A. duodenale, and 2 if >50% A. duodenale. In the ommunity level analyses, the hookworm speies variable represented low prevalene versus high prevalene shools. The models inluded all hild harateristis that differed between hookworm speies groups, or that we knew to be assoiated with iron status from previous analyses. 13 Adjusted mean values and standard deviations for iron status measures were alulated by analysis of variane. Whether and how to ontrol for the intensity of hookworm infetion presented a dilemma. Beause the blood loss aused by hookworm infetion is diretly proportional to the number of adult worms in the intestine, differenes in intensity (i.e. worm burdens) of A. duodenale and N. amerianus infetions ould onfound the omparison of iron status between individuals or ommunities infeted with the two speies. We measured intensity of infetion by faeal egg ounts, but these annot be diretly ompared between the two speies beause A. duodenale produes 2-3 times more eggs per adult worm than does N amerianus. Furthermore, there was not a simple way to 100

4 HOOKWORM SPECIES AND IRON STATUS 533 Table 1 Number of faeal samples diagnosed with hookworm infeaion by Kato-Katz method and faeal ulture method Kato-Katz result Negative Positive All Faeal ulture result Positive for Negative (6.3%) b A. duodenal! only (2.1%) N amerianus only (64.8%) A. duodenale + N. amerianus (26.9%) All 20 (3.8%) a 505 (96.2%) 525 a Column per ents. b Row per ents. The prevalene of N. amerianus infetion was uniformly high in all 10 shools surveyed, but the prevalene of A. duodenale varied greatly, from 2% to 88% (Figure 1). Four shools (Mihenzani, Tumbe, Msuka, and Mgogoni) were onsidered highly endemi for A. duodenale with prevalenes of 42-88%, while the remaining six shools had prevalenes below 18%. The four shools with high prevalene of A. duodenale were loated at opposite ends of the island, and did not share any obvious geographial harateristis (Figure 2). Beause A. duodenale KWALE VrtSHA CHOKOCHO NffOUBENI- MKHBCAN1 TIDJK USUKA MSOGOM nzdjbani S«NS JUU MApd 1t-13Apdi 1*-!8Aori l«-21afrt O-UWl 24-»M» * Etaokutog of rainy anon HOOKWORM SPECIES IDENTIFICATION SURVEY Pemba, United Republi o( Tanzania Figure 2 Map of Pemba Island, Tanzania, showing geographi loation of the 10 survey shools and the dates of the survey in eah shool transmission may exhibit seasonality, the dates of the survey in eah shool are inluded in Figure 2. The heavy rains began as the survey team moved from Chokoho to Ngombeni shool, and ontinued for the duration of the study. The four shools with high prevalene of A. duodenale were surveyed during the heavy rains, but so were Ngombeni, Kizimbani and Shengejuu whih had low prevalenes. Hookworm speies and individuals' iron status The harateristis of hildren with only N. amerianus infetion did not differ signifiantly from those infeted with A. duodenale (Table 2). The hildren were on average 10 years old, and about half were girls. About one-fourth of hildren had mirohaematuria, an indiator of urinary shistosomiasis, and around 60% were positive for Plasmodium faldparum. Trihuris trihiura and Asaris lumbrioides infetions were ommon. The prevalene and intensity of these infetions did not differ signifiantly between hookworm speies groups. The prevalene and intensity of hookworm infetion, assessed by the Kato-Katz method, did not differ signifiantly between the hookworm speies groups, but were signifiantly lower in hildren with negative faeal ultures (Table 2). The distribution of faeal egg ounts assessed by the Kato-Katz method was similar between the hookworm speies groups (Figure 3). By all indiators, the iron status of hildren infeted with A. duodenale was worse than those infeted with only N. amerianus, and haemoglobin and serum ferritin values beame progressively worse aording to the perentage of A. duodenale larvae found (Table 3). Overall there is a onsistent pattern of worse iron status in hildren with more A. duodenale infetion. The differenes between groups was most signifiant for serum ferritin, the indiator most speifi to iron defiieny. Hookworm speies and ommunity burden of iron defiieny and anaemia Compared to hildren in shools with low prevalene of A. duodenale, hildren in the four shools with high A duodenale prevalene were slightly older and had a higher prevalene of mirohaematuria (Table 4). The prevalene and intensity of T. trihiura or A. lumbrioides infeaion did not differ between hildren in the two groups of shools. However, the prevalene of hookworm infetion and hookworm faeal egg ounts were signifiantly higher in hildren in the shools with high A. duodenale prevalene. The differene in the hookworm faeal egg ounts was apparent both in the mean values (Table 4) and in the distribution of values (Figure 4). After adjusting for these differing harateristis between the groups, hildren in the high prevalene shools had signifiantly

5 534 INTERNATIONAL JOURNAL OF EPIDEMIOLOGY Table 2 Charateristis of study hildren by speies of hookworm infeaion determined by faeal ultures Charateristi Age (years) Sex (% girls) Mirohaematuna Malaria parasitaemia T. trihuria prevalene eggs/g faees A lumbrioides prevalene eggs/g faees Hookworms^ a prevalene eggs/g faees Negative ulture (n = 33) 10.4 ± 1.5 b 42.4% 24.2% 60.6% 93.9% 525 (73, 3763) d 72.7% 239 (6, 9072) 87.9% 258 (44, 1499) N. amerianus larvae only (n = J40) 10.5 ± % 23.4% 63.3% 97.0% 633 (117, 3439) 71.5% 240 (6, 9691) 96.8% 608 (102, 3607) A. duodenale* <50% of larvae (n=113) 11.0 ± % 24.3% 60.7% 96.4% 577 (108, 1856) 71.2% 233 (6, 9576) 98.2% 844 (188, 3790) Inludes 11 samples In whih only A duodenak larvae were found, and 141 samples In whih both spedes were found. b Mean ± SD An Indiation of urinary shlstosomiasis. d Geometri mean (-1 SD, +1 SD) Prevalene and intensity of hookworm Infeaion by Kato-Katz method 90 «70 60 g 50 ~ o 20 0 D Culture negative H N. amerianus only <50% A. duodenale >50% A. duodenale Hookworm eggs/g faees Kato-Katz method >50% of larvae (n = 39) 10.5 ± % 23.1% 54.1% 100.0% 602 (193, 1882) 76.9% 326 (10, ) 92.3% 420 (56, 3168) Figure 3 Distributions of hookworm faeal egg ounts by hookworm spedes ategory determined by larval ulture. Numbers of hildren in speies ategories, ulture negative, 33; N amerianus only, 340; <50% A duodenale, 113; *50% A. duodenale. 39. Distributions are not signifiantly different by x 2 test worse iron status by a variety of measures (Table 5). Haemoglobin and ferritin onentrations were lower, and protoporphyrin onentrations were higher in hildren from shools where A. duodenale was highly endemi. Disussion These results indiate that infetion with A. duodenale in hildren and heavy transmission of A. duodenale in shool ommunities is assoiated with a greater burden of iron-defiieny anaemia than infetion with N. amerianus. Previous laims of the greater pathogeniity of A. duodenale have been based on highly varying estimates of greater blood loss in A. duodenale infetion. To our knowledge, this is unique epidemiologial evidene that the previously measured differene in blood loss translates into a greater burden of hookworm-related anaemia in individuals and ommunities infeted with A. duodenale. The degree of iron-defiieny anaemia aused by hookworm infetion depends on the number of adult worms inhabiting the gut, the duration of infeaion, the iron stores and intake of the infeted individual ~ 24 and, as we have attempted to demonstrate, the speies of hookworm. Ideally, proof of speies

6 HOOKWORM SPECIES AND IRON STATUS 535 Table 3 Iron status of hookworm-infeted hildren aording to degree of A. duodenale infetion determined by faeal ulture A. duodenale <50% of larvae (n=113) N. amerianus larvae only (n = 340) Iron status Indiator* Haemoglobin (g/dl) Mean ± SDb 9 8 ± , ± (58, 162) (65, 183) 36,9 103 (62, 173) 14.5 (7.7, 27.5) (7.2, 25.4) (5.1, 18.1I) ± % <llb % <9 Protoporphyrln (umol/mol heme) ' Values are predlrted from analysis of vanane, adjusted for hookworm eggs/g faees determined by Kato-Katz method, age, helght-for-age Z sore, malaria parasitaemia, and sex. b Linear trend In Indiator by hookworm speaes ategory signifiant, P *; 0 05 Linear trend in indiator by hookworm speaes ategory signifiant, P < Table 4 Charateristis of study hildren in shools with low or high prevalene of A duodenale infetion determined by faeal ultures A. duodenale prevalene Charateristi Age (years) Sex (% girls) Mirohaematuria Malaria parasitaemia T trihiura prevalene eggs/g faees A, lumbrioides prevalene eggs/g faees Hookworms prevalene eggs/g faees 10.4 ± 1.7"' 48.5% 20.9% d 61.6% CJ Culture negative B N. amerianus only <50% A. duodenale >50% A. duodenale 60 Low, <20% High, >20% (6 shools, 308 hildren) (4 shools, 217 hildren) u 90 *80 g ± % 27.6% 62.3% 96.7% 637 (110, 3689)e 575 (123, 2692) 71.5% 234 (6, 9499) 259 (7, 9789) 94.8% b 486 (69, 3439) b 811 (180, 3652) 97.2% 72.6% f 50 i o Hookworm eggs/g faees Kato-Katz method Figure 4 Distributions of hookworm faeal egg ounts in hildren from shools with low (<20%) or high (*20%) prevalene of A. duodenale. Numbers of hildren in low and high prevalene groups are given in Table 4. Distributions are signifiantly different by %2 lest, P= % * Mean ± SD. b d Signifiantly different from high prevalene shools, P < An indiation of urinary shlstosomiasis. Signifiantly different from high prevalene shools, P < 0.10 Geometri mean (-1 SD, +1 SD). Prevalene and intensity of hookworm infeoion by Kato-Katz method. differenes in the impaa on iron status of individuals would be based on measurement of all of these fators. In reality, suh a study is nearly impossible to ondut. To aount for the duration of infetion, one would have to begin with an uninfeted individual, and follow longitudinally the ourse of infetion both in terms of speies and number of worms, the iron intake and bioavailability of the diet, and the iron status of the individual. Suh a study would illuminate our understanding of iron balane in hookworm infetion, the role of hookworm speies, and partiularly the adaptations that might our as haemoglobin levels beome autely low. However, the tehnial and ethial diffiulties of suh an observational study are likely prohibitive. In weighing the strength of the present epidemiologial evidene, it is useful to onsider eah of the four fators listed above. We measured intensity of infetion by faeal egg ounts, not by expulsion and enumeration of adult worms. The relationship between faeal ounts and number of worms is different for the two speies by a fator that is not preisely known. Adult female A. duodenale produe more eggs than do N. amerianus, and the male to female ratio of adult worms is higher for A. duodenale than for N. amerianus. This is problemati sine we would like to ompare the effet of speies at similar levels of worm burdens. One ould try to aount for the differenes by mathematially modelling the relationships, but 'the arithmeti Geometri mean (+1 SD, -1 SD) % >120 Ferritln (ug/1) Geometri mean (+1 SD, -1 SD)C % <12 C >50% of larvae (n = 39)

7 536 INTERNATIONAL JOURNAL OF EPIDEMIOLOGY Table 5 Iron status of Zanzibari hildren by prevalene of A duodenale in the shool A. Iron status Low, <20% Indiator* (6 shools, 308 hildren) Haemoglobin (g/dl) Mean ± SD 10.5 ± 1.5 b % <11 duodenale prevalene 58.9 % <9 Protoporphyrin ( imol/mol heme) Geometri mean 93 (55, 156) d (+1 SD, -1 SD) % > b Ferritin (jig/1) Geometri mean (+1 SD, -1 SD) % < (7.8, 28.0) 31.0 d High,>20% (4 shools, 217 hudren) 10.2 ± (65, 184) (6.9, 24.9) 48.4 ' Values are predited from analysis of variane, adjusted for hookworm eggs/g faees determined by Kato-Katz method, age, height-for-age Z sore, malaria parasitaemia, haematuna and sex. b Signifiantly different from high prevalene shools, P < 0 05 Signifiantly different from high prevalene shools, P < d Signifiantly different from high prevalene shools, P < relationships between hookworm load, egg ounts, and blood losses are diffiult to estimate reliably' 2 for a number of reasons. By simply inluding ategories of hookworm eggs/g faees in our analyses of the speies variables, we have ompared the iron status of individuals and ommunities within those ategories and thus have likely underestimated the impat of greater blood loss per worm with A. duodenale. Although an unbiased estimate of the speies differene based on numbers of worms is desirable, any mathematial model would be highly speulative, and this answer awaits a future study in whih worms are expelled and ounted. In the meantime, it is reasonable to infer that the true effet on iron status by the different speies is at least as great as our results would indiate. We did not measure duration of infetion nor iron intake and bioavailability. This does not threaten our inferenes unless duration of infetion or iron intake were different for hildren or shool ommunities in different speies ategories. We an think of no senario in whih the duration of infetion might have differed by speies, sine both speies likely have been endemi on Pemba Island for many years and hildren's risk of exposure with age is not known to differ for the two speies. It is somewhat more probable that dietary iron intake might be assoiated with speies in our ommunity level analyses. If hildren infeted with A. duodenale or shoolhildren in ommunities with high levels of A. duodenale infetion onsume less iron than their ounterparts infeted with N. amerianus, then our results ould be spurious. Laking dietary intake data, we annot prove that this is not the ase. However, we onsider it to be unlikely that in this environment where essentially all hildren are infeted with hookworms, the determinants of exposure to A. duodenale as opposed to N. amerianus would also be assoiated with poor diet. The two approahes we took to address the question of speies impat have omplementary strengths and weaknesses. The omparison of individuals by degree of A. duodenale infetion does not assoiate the speies variable with shool, an advantage beause shool ommunites may vary in level of poverty and thus diet. Rather hildren are ompared to other hildren, some of whom ome from the same shool ommunity. Our results from this analysis are also strengthened by the dosereponse pattern seen in haemoglobin and ferritin values by degree of A. duodenale infetion (Table 3). The omparison of hildren grouped by ommunity prevalene of A. duodenale infetion represents the pratial question that onfronts publi health planners. Are ommunities or areas where A. duodenale is highly endemi in greater need of intervention than ommunities where N. amerianus predominates? Based on our results, the answer to this question is 'Yes.' The strength of this analysis is that in reality the risk of A. duodenale infetion (or the fore of transmission of that parasite) is more harateristi of a ommunity than of an individual. The hildren in shool ommunities with high prevalene of A. duodenale who happened not to be infeted with that speies at the time of our survey, have likely experiened A. duodenale in the past or will do so in the future. The ommunity also represents the level at whih publi health deisions are made. The predominate hookworm speies being transmitted may be an appropriate means of haraterizing and targeting ommunities, but will rarely influene the ourse of treatment of an individual. Most importantly, the weight of our evidene is strengthened by the onsisteny of the findings from omplementary approahes to the problem. We onlude that, as predited from earlier linial studies, the speies of hookworm being transmitted in a ommunity influenes the burden of iron-defiieny anaemia in the ommunity. The full impliations of hookworm speies for the planning and implementation of interventions are yet to be determined. Our results suggest that where iron-defiieny anaemia is a publi health problem, hookworm ontrol is partiularly urgent if A. duodenale is the predominant speies. However, this should not be interpreted to mean that endemi N. amerianus infetion is of low priority. We and others have demonstrated previously that hookworms an be an important ause of anaemia in populations where N. amerianus predominates. 23 ' 24 Where anaemia is prevalent and hookworms are known to be endemi, lak of knowledge about hookworm speies should not impede interventions. A potentially important question is whether the optimal regimen for anthelminthi ontrol of hookworms in populations differs for the two spedes. Beause transmission of A. duodenale has a distintly seasonal pattern in some environments, it is possible that the effiay of publi health deworming regimens would depend on the seasonal timing of treatments. It is also possible that the rate of reinfetion following treatment differs for the two spedes, with impliations for the frequeny of treatments. Further epidemiologial researh is needed to answer these questions. Aknowledgements The authors thank the Helminth Control Team, espedally Mr Faki BF, for their exellent work in olleting these data.

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