The Potential Impact of Density Dependent Fecundity on the Use of the Faecal Egg Count Reduction Test for Detecting Drug Resistance in Human Hookworms

Introduction Soil transmitted helminth (STH) infections (Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale) contribute significantly to morbidity in humans in endemic countries. The major means of controlling these infections is by the periodic administration of anthelmintic drugs [1]. Given the lessons from livestock, where resistance to anthelmintics is widespread [2], the potential for anthelmintic resistance in human parasites has been recognized [3]. There are a number of differences in drug use patterns between the human and livestock fields which may aid in reducing the rate of resistance development in the former [4,5], however, the potential for resistance in human parasites is a significant issue [6]. It is clear that monitoring of drug resistance will be an important part of ensuring the success of current efforts to control STH infections. Although sensitive molecular tests are able to detect SNPs associated with benzimidazole resistance in livestock nematodes, they are still not available for human STHs. Phenotypic assays of worm eggs or larvae in vitro are useful for detection of resistance to some drug groups in livestock nematodes, however, their use with human STHs requires substantial validation and field testing. Hence, the faecal egg count reduction test (FECRT), in which egg output per gram of faeces (epg) is compared in individuals before and after drug treatment to estimate drug efficacy, is considered to be the most useful technique currently available for detecting the emergence of resistance in STH infections in humans. A recent study by Kopp et al. [7], in which dogs infected with the hookworm Ancylostoma caninum were treated with pyrantel, showed a poor relationship between drug efficacy and changes to epg. A mean drug efficacy of 71% in two dogs infected with an isolate showing low level resistance to the drug was associated with a 41% increase in epg. That is, egg output increased markedly despite the removal of a significant proportion of the adult worm burden by the drug. They suggested that this was due to an 1 October 2008 | Volume 2 | Issue 10 | e297 increase in egg production in surviving adult female worms after drug treatment due to the relaxation of density-dependent constraints on egg production. Density dependent fecundity has been demonstrated many times with Helminth parasites [reviewed by 8, 9]. The effect is seen as a decrease in egg output as the parasite burden increases. An additional feature typical of reports on density dependent fecundity is the presence of a greater degree of variation in fecundity at low worm burdens. However, the phenomenon is not associated with all Helminth infections, for example, several reports have indicated an absence of density dependent effects on the fecundity of Haemonchus contortus in sheep [10,11], while the evidence for Ostertagia circumcincta is equivocal [12], and Shaw and Moss [13] found no evidence of a density dependent effect on fecundity in Trichostrongylus tenuis in red grouse. In terms of human STHs, the density dependent effects have been reported for Ascaris lumbricoides [14–17], Necator americanus [18,19], and mixed N. americanus and Ancylostoma duodenale infections [20]. Anderson and Schad [20] noted that while the intrinsic fecundities of the two human hookworm species were similar, the density dependent constraints on egg production were more severe for N. americanus. For Trichuria trichiura the evidence is less clear. Bundy et al. [21] reported a strong density dependent effect on fecundity, while a later study [22] found that no significant association between fecundity and worm burden. The later report did however note that very high per capita fecundities were only observed at low worm burdens. Michael and Bundy [23] reported density dependent effects on fecundity in Trichuris muris. Density dependent effects on egg output have also been reported for the canine hookworm A. caninum [24,25]. It has been suggested that density dependent fecundity results from either competition for resources between parasites, or from immunological responses by the host [8,9], or from direct parasiteparasite interactions [26]. In the case of Strongyloides ratti infections in rats, the density dependent effects on the parasite’s establishment, survivorship, as well as fecundity, are mediated by host immune responses [27,28]. A number of reports have described the impact of density dependent fecundity on transmission dynamics [20,29], the rate of parasite reinfection following chemotherapy [30], and the spread of drug resistance [31]. We were interested in examining the effect that the phenomenon may have on the use of the FECRT to detect anthelmintic resistance in human STHs. If egg-laying by female hookworms is constrained by density dependent effects, the question arises as to whether this phenomenon is dynamic in nature if the worm density within a host is changed by, for example, a drug treatment. Survivors of a drug treatment will be present at a lower density after the treatment compared to before. Will the constraints on egg production be removed as the worm density decreases? The present study aimed to address this issue in terms of its potential to distort a FECRT by examining recent evidence on the response of the canine hookworm to drug treatment [7], and by examining an early N. americanus dataset describing the relationship between egg output and worm infection levels [18]. Methods Analysis of canine hookworm data Kopp et al. [7] examined the response of canine hookworms to pyrantel drug treatment. The hookworms were from a highly resistant isolate, and an isolate showing a low level of resistance. Infections were established in four dogs (two per worm isolate), and faecal egg counts were measured daily. Egg counts stabilised by day 24, and all dogs were treated with pyrantel. Egg counts were monitored for a further 6 days, and faeces was collected daily. The dogs were then euthanased and all worms present in the small intestine were counted. Total pretreatment worm counts were calculated as the sum of those present in the animal after drug treatment and those recovered from the daily feacal collections. Egg output (epg) data over the time course of the experiment was reported. For the present study we reanalysed the data from Kopp et al. [7] in terms of egg output per day (epday) per worm, rather than epg. Daily faecal weights had not been recorded so in order to do estimate daily egg output per dog we used a mean value for faeces output of dogs of similar size to those used in the Kopp et al. [7] study that were housed under identical conditions for an earlier experiment. This represents an approximation of the faeces output for each of the 4 study dogs; however the absolute amount of output is not crucial to our analysis, as long as it remained approximately constant before and after drug treatment. Kopp (pers. comm.) noted that there was no observable change in faecal output in each dog post treatment. Analysis of human hookworm data We examined the data of Hill [18] who reported egg output (epg, and epday per female) and worm burden (counting worms in faeces after treatment with carbon tetrachloride and oil of chenpodium) in human cases infected with N. americanus in Porto Rico in 1926. Complete data was obtained for 93 cases. Hill [18] noted a threshold of 500 females above which egg production per female was always low. We grouped the human cases separately: those above the 500 threshold which we term as ‘constrained’ in their egg output by density effects (n = 11), and the remainder of the cases with less than 500 worms (n = 82). We examined the effects on a FECRT if these ‘constrained’ worms are able to increase their egg output upon relaxation of the density dependent constraints by a drug treatment. We assumed that as a result of the removal of significant proportions of the adult worm burden (60–90%), all female worms in the ‘constrained’ group would be able to increase their egg output to the median value of those worms lying below the 500 female threshold. This represents an estimation of the post drug egg output as the worms in the 11 cases will most likely adopt a range of egg outputs after drug treatment as reflected by the great Author Summary Current efforts to control soil-transmitted helminth (