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This is the published version of an article in Parasitology, 106 (4)

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Quinnell, RJ, Slater, AF, Tighe, P, Walsh, EA, Keymer, AE and Pritchard, DI (1993) Reinfection with hookworm after chemotherapy in Papua New Guinea. Parasitology, 106 (4). 379 - 385. ISSN 0031-1820 http://dx.doi.org/10.1017/S0031182000067123

White Rose Research Online [email protected] 379 Reinfection with hookworm after chemotherapy in Papua New Guinea

R. J. QUINNELL1*, A. F. G. SLATER1!, P. TIGHE2, E. A. WALSH2, A. E. KEYMER1 andD. I. PRITCHARD2 1 Department of Zoology, South Parks Road, Oxford OX1 3PS 2 Department of Life Science, Nottingham University, Nottingham NG7 2RD

(Received 2 June 1992; revised 29 October 1992; accepted 30 October 1992)

SUMMARY Reinfection with hookworm (Necator americanus ) following chemotherapy was studied over 2 years in a rural village in Madang Province, Papua New Guinea. The prevalence of hookworm infection had returned to pre-treatment levels after 2 years, and the geometric mean hookworm burden had returned to 58 % of the pre-treatment value. The rate of acquisition of adult worms was independent of host age, and was estimated as a geometric mean of 2-9-3-3 worms/host/year (arithmetic mean 79-8-9 worms/host/year). There was significant predisposition to hookworm infection; the strength of this predisposition did not vary significantly between age or sex classes.

Key words: hookworm, Necator americanus, reinfection, predisposition, chemotherapy, Papua New Guinea.

1981). Hookworm is the commonest helminth in- INTRODUCTION fection in Papua New Guinea: in 1919-1923 the Hookworms (Necator americanus and Ancylostoma Australian Hookworm Campaign recorded a preva- duodenale) are among the most prevalent of all lence of 68% from a countrywide survey of more human infections. The distribution of these parasites than 46000 people (Sweet, 1924), while more recent extends throughout the wet tropics and subtropics, estimates give a prevalence of 74—90 % (Vines, 1970). and an estimated 1150 million people are infected Despite its commonness little is known about the (Bundy, 1990). A large number of studies of local population dynamics of the parasite, and hookworm epidemiology have been carried out, estimates of intensity have relied on the use of faecal especially in the 1920s and 1930s, when the Rocke- egg counts. In the only published reinfection study feller Foundation funded studies and control pro- from Papua New Guinea, Ashford & Barnish (1989) grammes in many countries (Ettling, 1990). How- found no predisposition to high or low hookworm ever, since this period the study of hookworm has egg count in children from a highland village. been neglected in comparison to that of other human The present study was carried out in a lowland helminths. In particular, there have been very few village on Karkar Island, Madang Province, Papua studies involving the use of anthelmintic-induced New Guinea. This study is part of a wider investi- worm expulsion to accurately determine worm gation of the epidemiology and immunology of N. burdens (Bundy, 1990). Worm expulsion has been americanus, the only species of hookworm found in used in recent studies in India and Zimbabwe the area; the study design and pre-treatment results (Anderson & Schad, 1985; Schad & Anderson, 1985; have been previously published (Pritchard et al. Bradley et al. 1992), but to date there has been only 1990). The present paper describes the extent and one community-based reinfection study with in- pattern of reinfection 1 and 2 years after treatment. tensity assessed by worm counts, that of Haswell- Elkins et al. (1988). Studies on hookworm in Papua New Guinea have concentrated on assessing its distribution and preva- MATERIALS AND METHODS lence, and its relationship to nutritional status, Fieldwork was carried out in the village of Kebasob, particularly anaemia (Zigas, 1973; Shield et al. Karkar Island, Papua New Guinea. Details of the study area, study design and methods used have * Present address: Department of Medical Parasitology, already been described (Pritchard et al. 1990). London School of Hygiene and Tropical Medicine, Briefly, an initial survey was carried out in July- Keppel St, London WC1E 7HT. f Present address: Picower Institute for Medical Re- August 1988. Faecal samples were obtained from search, 350 Community Drive, Manhasset, New York 202 people, who were then treated with the an- 11030, USA. thelmintic pyrantel pamoate (Combantrin, Pfizer

http://journals.cambridge.org Downloaded: 10 Sep 2009 IP address: 129.11.77.198 R. J. Quinnell and others 380 Ltd) at a dose of 10 mg/kg. All faecal output was were carried out according to Sokal & Rohlf (1981). collected for 48 h and expelled worms counted. Negative binomial distributions were fitted by an Later, anthelmintic treatment was offered to all iterative maximum-likelihood method (Elliott, villagers who had not been included in the study 1977). group: in total, an estimated 60% of the village population received treatment. In August 1989 faecal samples were collected from RESULTS as many as possible of the original sample. Five Prevalence of hookworm infection people were found to be infected with Ascaris lumbricoides and were treated with Combantrin; the The prevalence of hookworm infection before rest of the population was not treated. In July- chemotherapy, assessed by faecal egg counts, was August 1990 faecal samples were collected again, all 92-9%, which was reduced to an estimated 37% by people treated and counts of expelled worms per- chemotherapy. Prevalence had increased to 65-0% formed as in 1988. and then 80-7 % after 1 and 2 years reinfection (Fig. Only data from people who provided samples in 1). The prevalence assessed by worm recovery was all years are analysed: 140 people provided faecal 81-3% in 1988 and 82-7% in 1990. samples in all 3 years, and 75 people provided full The age-prevalence profiles of infection in all 3 post-treatment faecal output in both 1988 and 1990. years are shown in Fig. 2. After reinfection preva- The efficacy of anthelmintic treatment was as- lence did not increase with host age, and had sessed in a subsample of 25 people in 1988: the cure returned to pre-treatment levels in the younger age rate was 60 % and the reduction in arithmetic mean classes in 1990. Prevalence was very similar in male 2 faecal egg count 94% (Pritchard et al. 1990). and female hosts in all three years (Fig. 1; x — 1*17, Seventy-two h faecal collections were made from a 0-387and0053 in 1988,1989and 1990, D.F. = l.N.s. total of 20 worm-positive individuals in 1988 and in all cases). 1990. These people expelled a mean of 88 % of their worms during the first 48 h. Intensity of hookworm infection Faecal egg counts were performed according to a modified formalin—ether concentration technique After 2 years reinfection the geometric mean (95 % (Hall, 1981); results are presented as eggs per gram c.l.) worm burden was 4-8 (33-6-8) worms/host, of faeces (eggs/g). Worms were counted after 58% of the pre-treatment value of 8-3 (5-5-12-3) washing post-treatment faeces through two grades of worms/host. Corresponding arithmetic means were sieve (1 mm and 300 /u,m). The sediment was pre- 13-0 worms/host in 1990 and 25"3 worms/host in served in 10% formalin and worms were recovered 1988. Worm burden differed significantly between after careful searching by eye, counted and sexed. years, but there was no significant effect of host sex During the 3 years village meetings were held on worm burden. Although the percentage mean when the pathology of hookworm infection and its reinfection was lower in female than male hosts, 40 mode of transmission were explained to interested compared to 78%, there was no significant effect of villagers (in Melanesian pidgin). Leaflets were also host sex on the degree of reinfection (ANOVA: year, distributed advising people on latrine use and the F173 = 7-12, P<001; host sex, F1 73 = 2-01, N.S.; safe disposal of faeces. Ethical clearance for the study interaction, Fx 73 = 2-17, N.S.). was obtained from the Medical Advisory Committee The age-intensity profiles of infection were very of the Department of Health, Government of Papua different pre-treatment and after reinfection: mono- New Guinea. tonic pre-treatment and flat after reinfection (Fig. 3 and Pritchard et al. 1990). There was a highly significant correlation between worm burden and Statistical analysis host age in 1988 (r = 0-453, n = 75, P < 0-001) but The distributions of egg counts, worm burdens and not in 1990 (r = 0-090, n = 75, N.S.); these cor- host ages were positively skewed: these data were relation coefficients differed significantly (t = 2-39, analysed after logarithmic transformation. Predis- P < 005). position was analysed by non-parametric methods The geometric mean faecal egg counts in 1988, on untransformed data. Age and sex standardization 1989 and 1990 were 93, 23 and 45 eggs/g respectively of data (where indicated) was performed by z- (Fig. 4). The per capita egg production of female transformation within sex and age classes (3-8 years, worms was similar before treatment and after 2 years 9-13 years, 14-30 years and 31-60 years old in 1988). reinfection, geometric means (95 % c.l.) being 20 As there are problems in testing the homogeneity of (14-30) and 17 (11-27) eggs/g/female respectively rank correlation coefficients, the predisposition an- (paired i-test: D.F. = 45, t = 061, N.S.). In 1990, as alysis was also performed parametrically; data were in 1988, the sex ratio of hookworms was not converted to normal scores in order to normalize significantly different from unity, 490 males and 485 best the data within age-classes. Homogeneity tests females being recovered (x2 = 0-026, D.F. = 1, N.S.).

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100 r 100

80 80

c CD (0 CD m 40 40

20 20

0 1 2 Years after chemotherapy "0 1 2 Years after chemotherapy Fig. 1. The prevalence of hookworm infection pre- Fig. 4. The intensity of hookworm infection pre- treatment and at various times post-treatment in treatment and at various times post-treatment. Intensity Kebasob, Papua New Guinea. Prevalence was calculated was assessed as geometric mean faecal egg production from faecal egg counts. (#) Male hosts (n = 75); (O) (eggs/g). (•) Male hosts (« = 75); (O) female hosts female hosts (n = 65). (n = 65).

Table 1. Frequency distribution of hookworm 100 numbers per person in 1990: the aggregation parameter (k) and the goodness of fit to the 80 negative binomial distribution (%2 test) within age groups and sexes 60 Age class 2 40 (years) n k x 5-10 18 0-328 — 20 11-15 20 0-549 — 16-32 17 0-481 — 33 + 20 0-477 — 10 20 30 40 50 Total 75 0-447 4-22f Age (years) Males 39 0-451 418f 0-614 Fig. 2. Age-prevalence profiles of hookworm infection Females 36 2-24+ pre-treatment (1988) and 1 and 2 years post-treatment (1989 and 1990). Prevalence was calculated from faecal —, Insufficient degrees of freedom for x* test. egg counts. (•) 1988; (•) 1989; (•) 1990. Each-point f P > 005 (i.e. no significant departure from negative represents 16-35 hosts. binomial distribution). Table 2. Correlation coefficients between initial

30 r and reinfection hookworm egg counts (Kendall's rank correlation coefficient for raw data (T) and data standardized by age and sex (standardized T).)

n T Standardized T

1988 vs 1989 140 0-262*** 0-211*** 1988 vs 1990 140 0188*** 0-227*** I 10 1989 vs 1990 140 0-330*** 0-253***

*** P< 0-001.

The frequency distribution of hookworm burdens 10 20 30 40 Age (years) The frequency distribution of hookworm burdens in Fig. 3. Age-intensity profiles of hookworm infection 1990 was highly overdispersed, and could be de- pre-treatment (1988) and 2 years post-treatment (1990). scribed by a negative binomial distribution with Intensity was assessed as geometric mean ( + S.D.) worm aggregation parameter, k, of 0-447 (Table 1). There burden. (•) 1988; (•) 1990. Each point represents was little variation in k with host age or sex 17-20 hosts. (Table 1).

http://journals.cambridge.org Downloaded: 10 Sep 2009 IP address: 129.11.77.198 R. J. Quinnell and others 382 Table 3. Correlation coefficients between initial Kelly, 1966; Vines, 1970). The prevalence of and reinfection hookworm burdens hookworm infection in other villages on Karkar I. (T, Kendall's rank correlation coefficient; standardized T, was reported as 55—78% in the 1970s (Hornabrook, Kendall's rank correlation coefficient on age- and sex- Kelly & McMillan, 1975; Jones, 1976). Few standardized data; r, Pearson's correlation coefficient on population-based surveys of hookworm intensity data converted to normal scores.) have been carried out in Papua New Guinea; reported arithmetic mean intensities are typically n T Standardized T r <1000eggs/g (Ashford, Hall & Babona, 1981; Shield et al. 1981; Barnish & Ashford, 1989a, b), All hosts 75 0-311*** 0-301*** — though sometimes up to 3000 eggs/g (Bearup & Males 39 0-261* 0-277* 0-346* Lawrence, 1940). N. americanus is much the com- Females 36 0-422*** 0-354** 0-533*** monest species of hookworm in Papua New Guinea, 3-8 yearsf 18 0180 0-233 0-232 although A. duodenale has been reported in very 9-13 years 20 0-409* 0191 0-603** small numbers from a few coastal areas (Bearup & 14-30 years 17 019S 0-205 0-292 Lawrence, 1950). 31 + years 20 0-370* 0-389* 0-486* A rapid recovery of prevalence and intensity after chemotherapy is typical of helminth infections * P < 005; ** P < 001; **• P < 0001. t Age in 1988. (Anderson & May, 1985; Anderson & Medley, 1985). The percentage reinfection in the present study was relatively high compared to that reported for hook- Predisposition to hookworm infection worm infection in other areas (Table 4). The extent of reinfection is expected to be influenced by a There was a highly significant correlation between number of factors, including the proportion of the pre-treatment egg counts and those after 1 or 2 years population treated, the effectiveness of the anthel- reinfection (Table 2). Standardization of the data by mintic used and the basic reproductive rate of the host age and sex (see Materials and Methods section) parasite (Anderson & Medley, 1985). The relatively did not affect the significance of this correlation. high rate of reinfection recorded in Kebasob may be The correlation between pre- and post-treatment partly due to the relatively low proportion of the worm burdens was also highly significant (Table 3). village which was treated, around 60%. When hosts were divided into age and sex classes the In Kebasob people acquired a geometric mean of correlation was significant only in 9—13 year olds 4-8 adult worms after 2 years. The annual rate of and over 31-year-olds, and was stronger in female acquisition of adult worms can be calculated at than in male hosts. However, homogeneity tests on 2-9—3-3 worms/host/year from a simple immigration- Pearson's correlation coefficients between normal death model of infection (Anderson, 1986), assuming scores showed no significant differences between age 2 a constant mortality rate of 0-2-0-33/worm/year classes (x = 2-15, D.F. = 3, N.S.), and no difference (Hoagland & Schad, 1978). Such an annual rate between male and female hosts (t = 0-967, N.S.). would lead to an expected worm burden of Similar results were obtained with data analysed 10-15 worms/host in adults. Similar calculations after logarithmic transformation. using arithmetic means give an acquisition rate of 7-9-8-9 worms/hpst/year, and an expected adult worm burden of 27-40 worms/host. These burdens Other geohelminth infections are similar to, and well within the confidence limits A. lumbricoides and Trichuris trichiura were present of, the observed mean adult burdens pre-treatment at low prevalences in all 3 years. The prevalence of (Fig. 4 and Pritchard et al. 1990). This suggests that A. lumbricoides was 7, 0 and 3 % in 1988, 1989 and infection rates after chemotherapy were similar to 1990; all people infected with A. lumbricoides in 1989 those pre-treatment. were treated and are excluded from these figures. Reinfection with hookworm, measured by preva- The prevalence of T. trichiura was 1, 1 and 3 % lence or intensity, was independent of host age. A respectively. All prevalences were calculated from constant rate of acquisition of adult worms is faecal egg counts. Enterobius vermicularis was also expected to result in a monotonic age-intensity present at a prevalence (from worm counts) of 53 % profile, as was observed. Such monotonic profiles in 1988 and 7% in 1990. appear typical of hookworm infection both in Papua New Guinea (Barnish & Ashford, \989a,b) and elsewhere, although the number of such profiles based on worm counts is very small, particularly DISCUSSION from high-intensity areas. In contrast, reinfection The prevalence of hookworm infection in Kebasob is with A. lumbricoides and T. trichiura is typically most typical of that in lowland Papua New Guinea, with rapid in children, and age-intensity profiles are around 80—90 % of the population infected (Vines & convex (Bundy, 1990). These patterns suggest that

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Table 4. The relative intensity of reinfection (expressed as the percentage of pre-treatment intensity) with hookworm following treatment in different studies (The measure of intensity used was arithmetic mean egg count in all studies apart from that of Haswell-Elkins et al. (1988), who used arithmetic mean worm burden. Hookworm infections in Sri Lanka, Puerto Rico and Papua New Guinea were all N. americanus; those in India and Zimbabwe were a mixture of N. americanus and A. duodenale. In all studies anthelmintic efficacy was > 90%.)

Reinfection Time Study Country (%) (months) Sweet (1925) Sri Lanka 29 12 Hill (1926) Puerto Rico 10 12 Schad & Anderson (1985) India 30 21 Haswell-Elkins et al. (1988) India 35 11 Bradley & Chandiwana (1990) Zimbabwe 42 9 This study Papua New Guinea 43 12 This study Papua New Guinea 54 24

exposure to the infective stages of hookworm does sex. However, in the present study, although predis- not fall with increasing host age. Most hookworm position was detectable in only 2 of the 4 host age transmission, particularly of JV. americanus, is classes, there was no significant heterogeneity in thought to occur around defaecation sites, probably correlation coefficients between age or sex classes. during the process of defaecation (Chandler, 1929; Sample sizes were, however, small: if heterogeneity Schad, Nawalinski & Kochar, 1983). Infection by does exist, larger samples may be needed to detect it. this route may be less age-dependent than oral In contrast to the above studies, Ashford & Barnish infection, which depends on the ingestion of con- (1989) found no predisposition to hookworm in- taminated material such as food or soil. Practices fection in a highland village in Papua New Guinea. such as geophagia are far more widespread in However, this study was restricted to children, in children than adults (Bundy, 1988). whom predisposition was not detected in the present The lack of variation in the rate of acquisition of study nor those of Haswell-Elkins et al. (1988) and adult worms with age does not allow inferences to be Bradley & Chandiwana (1990). This lack of de- made about any possible role of acquired immunity. tectable predisposition may have a statistical rather Such a pattern may reflect either age-independent than biological explanation, as the lower intensity of exposure to infective stages or an increase in both infection in children will make predisposition harder exposure and immunity with age. Monotonic age- to detect (Keymer & Pagel, 1990). intensity profiles of helminth infection have been found even when there is significant resistance to A. lumbricoides and T. trichiura infections reinfection in adults (Quinnell, 1992). The frequency distribution of hookworm burdens A. lumbricoides and T. trichiura are typically much was, as expected, highly overdispersed, with an less common than hookworm in Papua New Guinea; aggregation parameter, k, of 0-447. This was similar Sweet (1924) recorded prevalences of 12 and 16% to that reported at the initial survey (0-370) and to respectively. The factors governing the distribution other reported values of 0-24 in southern India and abundance of A. lumbricoides and T. trichiura in (Haswell-Elkins et al. 1988), 0-346 in Zimbabwe Papua New Guinea are largely unknown, although (Bradley et al. 1992) and 0-63 in Bengal (Schad & both are more prevalent in the highlands (Vines, Anderson, 1985). 1970). Predisposition to hookworm infection has been In other villages on Karkar I. the prevalence of A. reported previously by Schad & Anderson (1985), lumbricoides has been reported as 21—57%, and that Haswell-Elkins et al. (1988) and Bradley & Chandi- of T. trichiura as 17-42% (Hornabrook et al. 1975; wana (1990). In the latter two studies the strength of Jones, 1976). The prevalence of both infections predisposition varied with both host age and sex, but reported in the present study was much lower, which the significance of this variation was not reported. may be due to better hygiene and a generally higher Predisposition to hookworm infection may be as- standard of living in Kebasob than in neighbouring sociated with variation in human behaviour, es- villages. It seems unlikely that either parasite, pecially choice of defaecation site, or variation in the especially T. trichiura, has a self-sustaining pop- strength of the immune response (Schad & Anderson, ulation in Kebasob, and it is possible that most cases 1985); both these factors may vary with host age and result from infection outside the village.

28-2

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