Hookworm Infection in Man

Hookworm Infection In Man THOMAS A. MILLER

* Jensen-Salsbery

Laboratories, Division of Burroughs Wellcome Co., Kansas City, Kansas, U.S.A.

I. Introduction.. ..................................

315 316 317 IV. Life Cycles and Infectivity.. .................................. 3 I9 321 A. Parasitic Development of Ancylostoma duodenale 323 B. Parasitic Development of Necator americanus ............. 325 C. Parasitic Development of Ancyfostoma ceyianicirm ....... ........................... 325 D. Auto-reinfection .......................................... ry Infection 325 E. Prenatal or Transmammary Infection .................. ... 326 F. Life Span of the Hookworms ............ V. Morbidity., ........................................... 327 321 A . Primary Morbidity in Acute Infection ...................... 330 B. Anaemia as a Primary Sign ........................... 331 C. Primary Morbidity in Chronic Infection ...................... 332 D . Secondary Morbidity in Chronic Infection ................... 338 ence of Morbidity VI. 339 ................................................ VII. 340 ................................................. 340 ................ Introduction ............................................................... 342 Classification of Trematodes.. .................... 344 Seasonal Studies of Metacercariae ................................. 347 .................. Seasonal Studies of Metacercariae in World Climatic Zones 349 ... ............................... A. Tropical .... 353 ........ .... VIII.B. Subtropical ........................... 354 C . Mid-latitude ......................................................... 354 D. Polar .................................................................. 354 E. Mountain ............................... 355 .................... F. Species Studied in more than one 356 D. Extreme Pathology in Fatal Acute Hookworm Infection .............................. General Conclusions, Metacercariae 357 E. Pathology in Organs Remote from the Hookworms .... Incidence and..intensity 1x.A. Immunology .............of...Occurrence ..................... ..................... ............................ 358 B. A.Principal and Auxiliary Hosts.................................... Diagnostic Immunology ................................................. 358 C. B.Invasion of Fishes by Cercariae .............................. Functional Protective Immunity ................ 360 Formationand of Visceral Metacercariae X.D. Cutaneous Larva .................................... Migrans _........._._.. .._................._..._ _...._.. 363 E. References Morphological .......Differences ................................. ......................,....... 365 11. General Life Cycles ................................................ 111. Distribution, Prevalence and Significance .........................

I. INTRODUCTION .................................

I . Seasonal in World Climate Zones .................. Hookworms areStudies parasites of mammals, being most frequent in primates, J . An Hypothesis for Seasonal Occurrence. a few species in other groups, including two carnivores and ungulates, with K. Experimental Studies ..... .............................. Seasonal Studies of was Adult Treniato * Part of this review compiled and written while the author was at the Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Scotland.

315

316

T H O M A S A. M I L L E R

aquatic mammals. Their natural distribution, which is determined primarily by the temperature requirements for development of their free-living stages, is equatorial, tropical or sub-tropical. As with most zoological classifications there has been considerable discussion over specific nomenclature (Looss, 1911; Lane, 1922; Yorke and Maplestone, 1926; Biocca, 1951, 1954; Rep, 1963, 1964) and frequent changes have been proposed in specific names, arrangement and grouping of species. The most recent review (Rep, 1963) proposed that the family Ancylostomidae comprises two sub-families, Ancylostominae and Bunostominae. The species to be mentioned in this and in a subsequent review (marked *), with their principal natural hosts, follow. ANCYLOSTOMINAE

Ancylostoma duodenale: man (and anthropoid apes?) * A . ceylanicum: man, dog, cat * A . hraziliense: dog, cat * A . caninum: dog * A . tubaeforme: cat

BUNOSTOMINAE

Necator americanus: man * Bunostomum trigonocephalum: sheep, goat *B. phlehotomum: cow * Gaigeria pachyscelis: sheep Consultation of an original reference (Rep, 1963) reveals more genera and species and much larger host lists. Many species described in that reference and some of the very large host lists have, however, been established on the basis of a few worms recovered from one or two individual animals. For instance, although for A . duodenale and its synonyms 24 species of host have been listed, man is probably the only true host. GENERAL LIFECYCLES The life cycles of all hookworm species are direct. Transport hosts may also be utilized by some species with carnivorous definitive hosts, to prolong the life of the infective larvae and to increase their chances of reaching their definitive host. There is therefore no intermediate host and the first two freeliving larval stages, derived from worm ova in the host's faeces, give rise to a third, free-living, infective stage in the environment of the host. This third larval stage commonly infects the host by skin penetration followed by migration and vascular or lymphatic transport to the lungs (Looss, 1911), whence the larvae reach the intestine via trachea and oesophagus. Little growth or development takes place before larvae enter the airways and the third moult (first parasitic moult) usually occurs after they leave the lungs. Fourth-stage larvae, with a provisional buccal capsule, are first found in the larynx, pharynx, oesophagus and intestine in a relatively short time after infection. In species in which life cycles have been determined experimentally, the length of the migration phase is fairly constant, occupying 11.

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about 7-15 % of the pre-patent period (i.e. of the interval between infection and first occurrence of eggs in the faeces). The last (fourth, or second parasitic) moult occurs in the intestine in which the fifth or adult stage matures, mates and reproduces. As a general principle, it appears that although lung migration is not essential for development of most hookworms, it offers the most convenient pathway with minimum effort to the intestine. After oral infection, in which larvae are first exposed to their proper host in the stomach (i.e. larvae ingested with food, administered experimentally in gelatin capsules or by stomach tube), many species mature in the intestine without first undertaking lung migration. However, if exposed to buccal, pharyngeal and oesophogeal epithelia for any length of time, some larvae penetrate and undergo lung migration before reaching the intestine. In abnormal hosts, the majority of larvae undergo lung migration, irrespective of route of administration, and subsequent worm development is slight or absent. The normal route of infection under natural conditions, and the proportions of larvae infecting the host by each route, are not known. Where both oral and percutaneous experimental infections have been successful, it would seem a reasonable supposition that natural infections occur by both routes, the relative importance of each being determined by host behaviour patterns and environmental influences. One notable exception to this outline life cycle is Uncinaria lucasi, the hookworm parasite of the fur seal. In this marine species chances of environmental infection are obviously extremely slender and the major route of infection to seal pups is by third-stage larvae in colostrum (Olsen, 1962). This route is also utilized by at least one other hookworm but only as a subsidiary mechanism. The length of time from infection to first appearance of hookworm eggs in the faeces of the host is, within certain limits, constant for each species of hookworm. The shortest prepatent periods (14-15 days) occur in the four hookworms of dogs and cats. The longest prepatent periods occur in the Bunostominae (e.g. the hookworms of ungulates and Necator americanus in man). Some reported variations in prepatent period may be attributed to experimental procedure, but various host influences (e.g. acquired and age resistance) interfere with worm metabolism and rate of development and have been shown to delay worm maturation. There is at present no solid evidence that strain, breed or race of host species can influence the success or rate of development of hookworms, or can alter their metabolism or pathogenicity. Species of host, however, has been demonstrated to influence hookworm development, from complete rejection of the parasite (Miller, 1966e) through reduced success in establishment (Miller, 1966a) to minor manifestations such as delayed development and restriction of worm growth (Miller, 1966d).

111. DISTRIBUTION, PREVALENCE AND SIGNIFICANCE Three species of hookworm occur in man, Ancylostoma duodenale, Necator americanus and Ancylostoma ceylanicum. The two principal hookworms,

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THOMAS A . MILLER

A. duodenale and N . americanus, have different geographical distributions. A. duodenale is sub-tropical and does not occur in the northern hemisphere above 52” latitude. Above 47” latitude its larvae require artificial shelter from the climate (e.g. mines or brick works) since the free-living stages require temperatures in excess of 22°C. The optimum temperatures for N . americanus are higher, in the tropical range of 25-28°C (Brumpt, 1958). The distributions of both species overlap, although one or other usually predominates. The true distribution of A . ceylanicum is not clear as until recently A . ceylanicum and Ancylostoma braziliense were considered to be the same species or host-induced strains of a single species (Rep, 1963). The consensus now differentiates two distinct species (Biocca, 1951; Biocca and Le Roux, 1958; Yokagawa and Hsien, 1961; Rep et al., 1968). Reports of the occurrence of these two species are confused and only the combined geographical distribution of both is at present described (Rep, 1963). Whenever A . ceylanicurn has been recovered from man, it has been found that it usually constituted only a small proportion of mixed hookworm burdens (Darling, 1924; Chandler, 1955; Areekul et al., 1970a; Chowdhury and Schad, 1972). The differing distributions of A . duodenale and N . americanus may be partly associated with ethnic and geographical origins of the population. For instance, in a series of surveys (Miller, 1970a) the occurrence of A . duodenale in East Africa appeared to be associated with communities or families containing members of Arab (i.e. Mediterranean) descent. This species of hookworm may also have been introduced to South America by people from the same area although Allison et al. (1974) presented evidence of pre-Columbia infection. N . americanus was almost certainly introduced to the North American continent by negro slaves (Hoeppli, 1970). Maps of the geographical distribution of the hookworms of man appear in most standard textbooks (Darling, 1922; Chandler, 1955; Faust and Russell, 1964) and in reviews (C.G.O., 1959; Rep, 1963). Estimates of the number of people infected with hookworms approximate one-quarter of the world’s population (CCTA-WHO, 1963; Stoll, 1962), exceeding at that time 600 million people. Stoll (1962), one of the principal authorities, stated “now that malaria is being pushed back, hookworm remains the great infection of mankind”. Le Riche (1967) showed that the world prevalence of hookworm infection in man exceeds the prevalence of almost all other helminthic infections and of other individual diseases, with the exception of only the common cold. The global significance of hookworm disease of man has thus remained essentially unchanged for more than half a century since 1911, when Boycott stated, “Taking the world as a whole, with the possible exception of the malaria organisms, ankylostoma is . . . responsible for more unhappiness and inefficiency than any other parasite . . .”. This is probably an over simplification of the situation as great improvements have been achieved in the developed countries in which hookworm disease of man, but not necessarily hookworm infection, have been almost eradicated. However, improvements achieved in developing countries are probably more than nullified by other changes, e.g. population expansion,

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which by analogy with the well-established association in the veterinary context between epizootic parasitism and population density, often lead to a resurgence of parasitic infection and disease. Views on the global significance of hookworm infection in man vary; Ansari (1971), for instance, omitted hookworm from a list of what he considered to be the five most important parasitic diseases of man. Additional reviews of geographical incidence and intensity of hookworm infections in man may be found in Erhardt and Schulze (1961). IV. LIFE CYCLESAND INFECTIVITY Information on route of infection, infectivity and parasitic life cycles of A . duodenale and N . americanus is confusing and seriously deficient. Deficiencies are attributable principally to the relative scarcity of suitable alternative hosts, in which critical experiments may be conducted, and to the natural reluctance to perform experiments on man. Both A . duodenale and N. americanus can infect various non-human hosts. Although mature worms have been reported in a number of wild, domestic and laboratory mammals, experimental larval infectivities have been very low, seldom exceeding 5 % and usually being less than 1 % (Kamati, 1943; Yoshida and Okano, 1959; Yoshida et al., 1960; Nagahana et al., 1962b; Tanabe, 1962; Yoshida and Fukutome, 1964, 1967; Miller, 1966a). In the few naturally occurring infections in mammals other than man, the numbers of worms recovered have also been small (Lane, 1917; Baylis and Daubney, 1922; Stiles and Hassall, 1929; McClure, 1932; Cameron and Myers, 1960). Small laboratory animals are completely or relatively unsatisfactory hosts since hookworms do not complete their development (Hung, 1926; Nakajima, 1931; Schwartz and Alicata, 1934; Sakoda, 1954; Sawada et al., 1954a; Nagahana et al., 1962a), or only a very small proportion reach adult stage (Sen and Seth, 1967; Yoshida and Fukutome, 1967). Only chimpanzee appear to offer a suitable alternative host since infectivities in the range of 30-50% for A . duodenale (Miller, 1968) and N . americanus (Orihel, 1971) were recorded. However, experimental work in chimpanzee and other apes is prohibitively expensive and in most situations ethically questionable. In spite of these difficulties, there is sufficient information from results of a few experimental infections of man (conducted mainly in Japan), supported by some results in experimental animals, to convey a general impression on the parasitic life cycles of the human hookworms. Failure of A . duodenale and N . americanus to mature in laboratory hosts has been shown on occasions to be associated with persistence of dormant but viable infective larvae in the tissues of that host. The abnormal host may then serve as a paratenic host. Although a complete life cycle through paratenic and definitive hosts has been proven in only two animal hookworms (Miller, 1970b) there are indications that other hookworms, including A . duodenale but not N . americanus (Soh, 1958), may have the potential capability of utilizing this type of predator-prey life cycle. The controversy on route of infection and infectivity has continued for some time. Boycott (191 l), Kendrick (1934), Brumpt (1958), Yamashita

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(1958), Nakamura (1960) and Akimoto (1966) achieved successful infections of man with A . duodenale (up to 60% take) by the percutaneous route. Attempted infections by the same route failed (Boycott, 1911;Yoshida et al., 1958; Sagitani, 1960) or gave poor results (Yanagisawa and Mizuno, 1961). In Yoshida et al.’s (1958) experiments, larvae penetrated the skin successfully but the infection failed to reach patency and Yanagisawa’s and Akimoto’s takes after percutaneous infection were equivalent to less than 1 % and 0-11 % respectively. Oral infections of man with A . duodenale larvae have usually proved successful (Boycott, 1911 ; Yamashita, 1958; Yoshida et al., 1958; Koike, 1960; Sagitani, 1960; Yanagisawa and Mizuno, 1961; Yamamoto, 1965) with resultant takes of up to 75 %. It would seem that the variability of the results and perhaps many of the failures may be attributable to differences in experimental procedure and infectivity of larvae, rather than to inherent differences between the two routes. In experiments in man infectivities were measured by worm recovery after anthelmintic medication. This technique represents a potential source of variation, particularly when few larvae are given and worm burdens are small. When necropsy and worm recovery was possible after experimental infection of laboratory animals with A . duodenale, there was no clear indication of which route was superior. The findings after experimental infection of man with N . americanus have been more consistent. Usually, percutaneous infection was successful and oral infection unsuccessful. Experimental oral infections by Kendrick (1934), Fujiita et al. (1957), Yamashita (1958), Yoshida et al. (1958) and Yanagisawa and Mizuno (1961) failed, and Koike (1960) was successful in only one of three attempts. The results of experiments by Mizuno and Ito (1963) and Nagahana et al. (1963) indicated that if during oral infection the larvae were encouraged (by prolonged contact) to penetrate the buccal epithelium infection was successful. Percutaneous infections were successfully established in man by Smith (1905), Beaver (1955), Yoshida et al. (1958), Nakamura (1960), Yanagisawa and Mizuno (1961), Yamamoto (1965), Fujiita et a/. (1957), Yamashita (1958) and Akimoto (1966). A few takes exceeded 40%, although most were less than 1 %. These takes are lower than those reported for other canine and feline hookworms in their normal hosts. In experimental infections of other mammals (i.e. abnormal hosts) with N . americanus, percutaneous infection was irregularly successful with very low takes (Kamati, 1943; Yoshida et a/., 1960; Yoshida and Fukutome, 1967; Miller, 1966a; Sen and Seth, 1967); oral infection failed (Nagahana et al., 1962a). Techniques of oral infection may account for apparent contradictions in the literature since administering larvae in gelatin capsules or by stomach tube is very different from oral infection by pipetting larvae into the mouth of the experimental animal or man. Gelatin capsule and stomach tube infections were uniformly unsuccessful in dogs (i.e. N . americanus larvae did not leave the alimentary canal to migrate into the body), but pipetting was often partly successful since larvae penetrated the buccal and oesophageal epithelia, reached the intestine after lung migration, and developed in the intestine (Nagahana et al., 1962b, 1963). Larvae of N . americanus therefore seem to be unable, or are not stimulated, to penetrate gastric or intestinal

HOOKWORM INFECTION I N MAN

32 1

epithelia, and further development is unlikely where larvae are first exposed to the host in these organs. N . americanus larvae that penetrate the epithelia, whether skin or buccal and pharyngeal epithelia, undergo an essential lung migration and are conditioned for further development (Sekine, 1965; Ishikawa, 1966). Little is known about the infectivity of A . ceylanicum to man although successful percutaneous infections have been described (Maplestone, 1933 ; Haydon and Bearup, 1963; Wijers and Smit, 1966; Bearup, 1967; Yoshida et al., 1972). Figures for infectivity (i.e. percentage take) are available in only the last report in which oral infection of volunteers was reported to be more successful (40-60 % take) than percutaneous infection (0-5 % take). In dogs infected with A. ceylanicum by the percutaneous route takes of 5-30% were achieved (Rep, 1966a). A.

PARASITIC DEVELOPMENT OF A . DUODENALE

Difficulties encountered when attempting to elucidate route of infection and infectivity in man are also met when attempting to define endogenous parasitic life cycles. Most of this information has therefore been obtained from the results of experimental infections of abnormal hosts, since the timing of events between larvae entering the human body and first appearance of eggs in the faeces must remain largely in the realm of speculation. Some supporting inferences may also be drawn from the chronology of clinical signs and symptoms. Although infectivity or percentage takes in experimental animals may be less than those reported in man, prepatent periods are similar. It seems reasonable to assume that larval migration and worm development are likely to progress at similar rates in human and in experimental animal hosts. Prepatent periods reported after experimental infection of man with A. duodenale have varied. A uniform prepatent period of 38-41 days was observed by Brumpt (1952) after experimental infections of 50 people. Looss (1911) reported prepatent periods in man of 45-74 days, Yoshida et al. (1958) 43-162 days, Koike (1960) 88 days and Akimoto (1966) 68-69 days. Some of the higher figures were associated with very light infections or with delay in faecal examination. A strain of A . duodenale from India has recently been shown to possess an extended variable prepatent period. Larvae acquired during the dry season do not mature so that eggs are not excreted until the next monsoon season (Schad et al., 1973). In addition to being observed in indigenous populations, this phenomenon occurred in an infected individual who travelled round the world to an entirely different seasonal area and also in a second person infected with this strain (Nawalinski and Schad, 1974). These findings, although based on only a few observations, suggest some control mechanism in the parasite or host in the form of a biological clock or, less likely, in an environmental sensor. In experimental animals the prepatent periods of other strains of A. duodenale were more uniform and were similar to lower figures recorded in man. The prepatent period in four chimpanzees was 34 days (Miller, 1968).

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T H O M A S A . MILLER

Mature, female worms containing ova were recovered at necropsy of monkeys and pups on the 31st day (Miller, unpublished work) and from pups on the 28th day (Yoshida and Okano, 1959). It seems unlikely that these experimental animals were more suitable hosts for A. duodenale than man since the percentages of worms recovered from monkeys and pups were low. The shortest prepatent period of A . duodenale (i.e. eggs first detected in faeces) is therefore about 35-40 days. Mature female worms, that have not yet commenced egg laying, may be present from about 31 days. The chronology of A . duodenale larval and worm development has been investigated in various hosts (Table 1) and a fairly uniform consensus is TABLE1 Development of A. duodenale in fhe intestine of some experimental mammals

Stage of worm

Interstadial moult

3

3 14 314

4

Mature

Pups

Monkeys" Chimpanzee

-23 23

-24 18-24

23-30 2641 31-43 28-36

18-28 34-39 31-40

11-

28-35 12-35'

References Okamoto (1961) Nagahana and Yoshida (1965) Higo (1961), Okamoto

0-3b 3 4 3-12

415

5 (immature) 5 (maturing)

Days after infection of

(1961)

Okamoto (1961) Nagahana and Yoshida (1965) 34

Miller (1968) Yoshida and Okano (1959) Nagahana and Yoshida (1965) Mehrotra et al. (1 964)

a Where references are not quoted, results are from experiments (unpublished work) with monkeys and pups by the author. Monkeys included several species of the genera Cercopithecus and Papio. Third-stage larvae in mucosa of intestine. Prepatent periods as short as 12 days (i.e. eggs first found in faeces) in dogs in A . caninum-enzootic areas suggest accidental and prior infection with this canine hookworm.

apparent. After 2-3 days in the intestinal mucosa (i.e. after oral infection), the third-stage larvae emerge into the lumen and undergo the third moult almost immediately. The chronology of this early phase after percutaneous infection has not been documented in experimental animals. Brumpt (1952,

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1958) concluded, from observation of the development of clinical signs in man, that A . duodenale larvae are in the lungs on the 3rd day, in the pharynx on the 4th day and in the duodenum on the 8th day after experimental percutaneous infection. Fourth-stage larvae have been recovered from the intestine of experimental animals up to 24 days after infection and the fourth moult occurs at 18-24 days. Immature fifth stage or adult worms are present until about 28 days, after which genital development progresses rapidly and by 34-40 days almost all worms are mature and female worms pass eggs. An unusual deviation in the model hookworm life cycle was observed by Nagahana and Yoshida (1965) after infection of pups with A . duodenale. Thirdstage larvae administered by stomach tube burrowed into the intestinal mucosa and remained there for 2-3 days, without undergoing pulmonary migration. If administered by subcutaneous or percutaneous routes, larvae underwent lung migration but without growing, and on reaching the intestine they entered the mucosa from which both orally and percutaneously administered larvae emerged as fourth stage. This is different from the model hookworm behaviour as typified by A . caninum in dogs, where growth and development occurs during lung migration and only larvae given by stomach tube or gelatin capsule enter the mucosa of the intestine (Matsusaki, 1950). B.

PARASITIC DEVELOPMENT OF N . AMERICAh’US

Prepatent periods reported after N . americanus infection of man have varied widely. The lower limit is about 44-56 days (Smith, 1904; Looss, 1911 ; Beaver, 1955; Nagahana et al., 1963). Periods of 70-100 days have also been described (Yoshida et al., 1958; Nakamura, 1960; Nagahana et a/., 1963). Longer prepatent periods were often associated with very light infections (Yoshida et al., 1958). In experimental-animals prepatent periods as short as 35 days have been observed (Sen and Seth, 1967) although figures have more frequently been about 50-60 days. Some longer periods, of similar duration to those observed in man, have also been recorded in laboratory animals (Nagahana et al., 1962b; Miller, 1966a; Yoshida and Fukutome, 1967). Development of N . americanus in both man and experimental animals appears therefore to be slower than A . duodenale. The chronological development of clinical signs and symptoms after experimental infection of man with N . arnericanus, as potential indicators of worm location, has been described by Smith (1904). The cutaneous reaction to infection subsided in 7-12 days. This was succeeded by upper respiratory signs that indicated larvae were in the lungs from the 7th day. By the 10th day, when the first larvae were reaching the intestine, the predominant symptoms were abdominal. From the results of experiments in laboratory animals (Table 2) it appears that after percutaneous infection, N . americanus third-stage larvae tend to remain in the dermis or other migratory locations and lungs longer than A . duodenale, so that the third moult is delayed about 6 days. This moult occurs in the intestine. Fourth-stage larvae may be recovered from the intestine until the 35th day, by which time most larvae have completed the fourth moult (20-23 days). Immature adults without

TABLE 2 Development of N. americanus in some experimental mammals

Days after infection at which stages recovered ~

Host Rabbit Guinea pig

3

3/4 moult

4

4/5 moult

0-8" 4-13b

11-?

11-32

24-32

5 (immature) 5 (maturing)

24-28

14-18

2 1-27

Pups

13

26

Pups Pups Monkeys Chimpanzee and Patas Monkeys

0-2" 3-10b

0-4"

3-10b

63

9-?

Hamster

Pups

48

Mature

8-10

9-22

20-22

8-1 3

8-26

21-27

2140

8-26

19-26

2143

?-35

17-35

1740

35-56 3145

71 51-56

References Yoshida and Fukutome (1967) Schwartz and Alicata (1934) Sen and Seth (1967), Sen (1972) Yoshida et al. (1960)

47-55

Nagahana et al. (1962b) Tanabe (1962)

31-56

52-56

Miller (1966a)

34-44

?-60 42-54

Miller' Orihel (1971) ~~

In skin or buccal epithelium. In lungs. ' In this unpublished work, monkeys included species of the genera Galago, Cercopithecus, Erytlirocebus and Papio. a

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genital development are present from the 20th to 40th day, genital developmen commences about 31-35 days and mature worms, including ovipositing females, are present by 42-60 days. PARASITIC DEVELOPMENT OF A . C E Y L A N I C U M

C.

Prepatent periods for A . ceylanicum in man have been reported at 21, 24 and 35 days by Wijers and Smit (1966), Bearup (1967) and Maplestone (1933) respectively. Yoshida et al. (1972) recorded prepatent periods of 18 to 26 days in six volunteers. In experimental infections of dogs and cats with this hookworm, Rep (1965) and Yoshida (1968) observed shorter prepatent periods of 14-17 days, similar to the typical periods in dogs after infection with their specific hookworms, A . caninurn, A . braziliense and U . stenocephala (Miller, 1964, 1966d,e, 1971). There is no information on the chronology of larval and worm development after A . ceylanicum infection of man. In experimental animals these temporal constants were found to be similar to those observed in A . braziliense and A . caninurn (Yoshida, 1968). D . AUTO-REINFECTION

An unusual event in the parasitic life cycles of A . duodetrale, A . ceylanicum (or A . braziliense) and N . arnericanus (Whipple, 1909; Bonne, 1937, 1942; Lie Kian Joe and Tan Kok Siang, 1956; Biaggi et al., 1957; Pagan and de Vega, 1963; Piza and Biaggi, 1963) is the occurrence of adult worms in submucosal closed cysts in the human intestine (usually jejunum). These cysts were enclosed by an outer inflammatory zone and were sometimes associated with local peritonitis. The central space, filled with blood, contained an adult hookworm. The cysts contained eggs, first- and second-stage but no third-stage larvae. The missing infective third-stage larvae could be expected to leave the cyst and migrate into the mucosa, subsequently to emerge into the intestine and complete an auto-reinfection life cycle without leaving the host. Although records of these cysts are few, they have been described in a relatively large proportion of the necropsy reports in which hookworms were the cause of death (e.g. more than 10 % of Whipple’s cases). These observations indicate an opportunistic (or evolutionary ?) trend, circumventing environmental hazards, advantageous to the hookworm. E.

PRENATAL OR TRANSMAMMARY INFECTION

“Prenatal” infection with many migratory helminths, including A . caninurn in dogs, has been reported. It has been shown, however, that with respect to A . caninurn, “transmammary” infection is the more appropriate term (Miller, 1970~).Since parasitic migratory life cycles of most hookworms are similar, it might be hypothesized that larvae of human hookworms would occasionally occur in colostrum and milk to cause apparent “prenatal” infection of children. Howard (1917) and de Langen (1923) reported findings which strongly suggested that this had occurred. In both cases, the mothers

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were exposed to infection in a hookworm-endemic area during pregnancy and hookworm eggs were found in faeces reputed to be from their children 1-2 weeks after birth. In only one case (Howard, 1917) was confirmation provided by repeated faecal examinations. Attempts were not made to recover worms, nor were species ascertained. It is surprising that infection was detected by faecal examination at such an early age. “Prenatally” acquired (via the colostrum and milk) canine hookworm larvae have been shown to be third stage and eggs are not usually demonstrable after birth sooner than the minimal prepatent period. The records of alleged human “prenatal” infection suggest that worms had either entered the foetus and commenced development 2-8 weeks before parturition or that fourth- or fifth-stage worms had been transferred via the colostrum. Both explanations are unlikely. It is, nevertheless, accepted that hookworm infection is uncommon in children under 1 year and extremely rare under 6 months. Braun (1965) described a severe infection (species unspecified) with a prepatent period of 50 days after birth and clinical and haematological signs at 26 days. The chronology of this case could be explained only by colostral or other immediate environmental neonatal infection. Similarly, Hollander et al. (1973) described a massive infection with N . americanus with signs of intestinal hookworms (typical acute hookworm stools) in an 8-day-old baby followed by eggs in the faeces when 32 days old. An hypothesis of prenatal or transmammary infection is unnecessary in this case since the mother had attempted to dispose of her newborn baby in a pit latrine in a hookworm endemic area. The short prepatent period is, however, remarkable for this species. F.

LIFE SPAN OF THE HOOKWORMS

Ancylostoma duodenale and N . americanus are comparatively long-lived. Kendrick (1934) recorded maximum worm egg output 12-18 months after experimental infection with A . duodenale, and a maximum life span of 6-7 years. He also showed absolute longevity of N . americanus to be similar (5-6 years). Boycott (191 1) reported persistent A. duodenale infections up to 6 years. Palmer (1955) described an infection of N . americanus that persisted for 15 years in the absence of reinfection. In spite of these extreme figures for absolute longevity, most reports describe considerable reductions in worm burdens (i.e. 70-80 %) in the absence of reinfection within 1-2 years (Mhaskar, 1920; Chandler, 1929) or 2-3 years (Kendrick, 1934). Only in Kendrick‘s (1934) and Palmer’s (1955) experiments is the species known. In Mhaskar’s (1920) and Chandler’s (1925) experiments the initial worm population was of various and unknown ages since their subjects had previously been naturally, rather than experimentally, infected. Longevity of individual worms may vary depending on whether reinfection or superinfection occurrs. If immunologically mediated self-cure or expulsive phenomena occur in the manhookworm relationship, as in other systems, continuing environmental reinfection could be expected to result in a worm population turnover in the dynamic interaction of host and parasite.

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In spite of extreme figures for absolute longevity, it is probably correct to assume that significant or pathogenic longevity, in the absence of reinfection and other contributing factors (i.e. dietary deficiencies, stress), should be measured in months (e.g. up to 30) rather than in years. Saint-Martin and Dussault (1957) reported a severe case of anaemia due to A . duodenale infection, for which treatment was not sought for 18 months after infection, during which time the subject had not been exposed to reinfection. Boycott and Haldane (1903) described subjects in which moderate to heavy infections with A . duodenale, accompanied by severe anaemia, persisted for 23 to 4 years after the last known infection. There is no information on the longevity of A . ceylanicum in man. In hookworm-endemic areas, the maintenance from year to year of reasonably constant burdens in the population would indicate that there is a continuous turnover in the hookworms comprising these burdens (Hsieh, 1970; Miller, 1970a) and would also lead one to suggest that there must be some intrinsic host factor that limits the size of these burdens (e.g. immunity). V. MORBIDITY In man, the signs and symptoms of hookworm infection may conveniently be separated into primary and secondary phenomena. Primary signs and symptoms are those associated specifically with the migration of infective larvae and the presence of adult worms. Secondary signs and symptoms are consequences of physiological, biochemical and haematological disturbances that follow the persistence of intestinal infection. Secondary signs and symptoms constitute what is more commonly termed “hookworm disease”. However, this term describes a spectrum of disorders that are so variable, and often associated with SQ many concomitant factors and influences, not a direct consequence of the presence of intestinal hookworm infection, that it would perhaps be better if the term were to fall into disuse. References to, and descriptions of the consequences of hookworm infection are so numerous that it will suffice to list but a few of the more complete articles and monographs (Ashford, 1900; Ashford et al., 1933a; Stiles, 1912; Darling, 1922; Lane, 1932; Cruz and de Mello, 1948; Arora, 1951; Brumpt, 1952, 1958; Borrero et al., 1961; Foy and Nelson, 1963; Woodruff, 1965; Ball, 1966; Matsusaki, 1966; Vieira, 1970). The review by Matsusaki (1966) is particularly useful since it catalogues in detail for the first time in English the voluminous Japanese language literature, comprising more than 600 articles. Reviews are also incorporated in most texts on medical helminthology and tropical diseases (Chandler, 1955; Dammin, 1962; Faust and Russell, 1964; Manson-Bahr, 1965) the first of which, the Ebers Papyrus, was written about 3500 years ago (Scheuthauer, 1881; Hoeppli, 1959). A.

PRIMARY MORBIDITY IN ACUTE INFECTION

The signs and symptoms of infection and their order of appearance are closely related to chronology of migration and development of the hookworms. There do not appear to be significant differences between the three

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species, A . duodenale, N . americanus and A . ceylanicum, except in relation to time factors in their endogenous life cycles. The first symptom after cutaneous infection is an immediate stinging sensation followed by dermatitis. These appear to be consequences of reactions that accompany penetration of larvae, secretions of migrating larvae and ensuing inflammation. The severity and persistence of skin reactions seem to vary whether following primary or reinfection (Brumpt, 1952). Immediate and delayed hypersensitive reactions on reinfection increase and extend the severity. The skin reactions vary from erythematous papules lasting 7-10 days on primary infection to severe papulation, vesiculation and generalized oedema of the area (usually a limb), with enlargement of the regional lymph glands that may persist more than 2 weeks after reinfection. Secondary bacterial infections consequent of scratching are said to be common. However, it is not necessary to incriminate scratching since hookworm larvae are invariably contaminated internally and externally with faecal and environmental bacteria. That bacteria intensify and extend inflammatory reactions was confirmed by the surprising levity and brevity of reactions after penetration of sterile axenically-cultivated larvae of N . americanus (Miller, 1975). After penetrating the skin, larvae of N . americanus appear to leave subcutaneous tissues rapidly (Smith, 1904). Infections with human hookworms rarely produce serpentine tracks. This is in marked contrast to the consequences of infection with some abnormal species of hookworm. A . ceylanicum, that very close relative of A . braziliense, does not cause creeping eruption in man (Bearup, 1967); further evidence that these two species are different. The sequential signs and symptoms of further larval migration are associated with the presence of larvae in the lungs, and with their escape from the pulmonary circulation to the airspaces. Vague malaise and transient fevers have been recorded frequently 2-4 weeks after infection (Smith, 1904; Ashford et al., 1933b; Myhre and Wallace, 1956; Dammin, 1962); these have been compared to influenza (Smith, 1904). Nevertheless, fevers are neither a constant nor severe sign (Hodes and Keefer, 1945; Brumpt, 1952). It is not clear if occasional fevers during the pulmonary phase are associated with antigens and secretions of the larvae. Micro-organisms ingested by first-stage and second-stage larvae, carried to the lungs and there released may also be responsible. Hookworm-associated bacterial pulmonary complications do not seem to be important in man and animals; therefore it would seem that few micro-organisms are released at this time and/or these microorganisms are not primary pathogens or not pathogenic in the numbers available. Micro-organisms in the intestine of the infective larva are an intrinsic part of the larva and every man and animal that receives a natural infection of hookworm also receives a small inoculum of micro-organisms. Absence of severe pulmonary signs is surprising, since infection of dogs with large numbers of A . caninum larvae produced fatal pulmonary haemorrhage (Miller, 1971). Failure to record similar signs and symptoms after infection of man with A . duodenale and N . americanus is almost certainly related to the small numbers of larvae administered in experimental infections. Brumpt (1952) mentioned retrosternal pain during the 2nd week after

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infection with A . duodenale but was unable to detect pulmonary signs or radiological evidence. He therefore proposed dismissing hookworm infection as a potential cause of Loffler’s syndrome or pulmonary eosinophilia. Accidental infections of man with large numbers of larvae of the canine hookworm ( A . caninum) did cause chest pains and detectable temporary pulmonary radiological changes (author’s observations, see below). Although the pulmonary migratory phase of human hookworm infection has not been associated with significant clinical signs and symptoms, the ensuing stage, during which larvae traverse the upper respiratory tract, has frequently been documented as a time of well-defined primary signs and symptoms (Hodes and Keefer, 1945; Duvoir and Brumpt, 1944; Dammin, 1962; Hackett et al., 1964). Upper respiratory signs and symptoms after infection with A . duodenale include coryza, pharyngitis and laryngitis, sensation of obstruction in the throat and pain when speaking and swallowing. These start as early as 3 days and persist for 2 weeks after infection (Ashford et al., 1933b; Kendrick, 1934; Laederich et al., 1944; Myhre and Wallace, 1956). At this time Kendrick (1934) recovered larvae from the sputum of experimentally infected subjects. Brumpt (1952) suggested that persistence of upper respiratory signs and symptoms (for as long as 3 weeks) may be caused by aberrant larvae wandering in the epithelium of the larynx and pharynx. Although migration and development of N . americanus appears to be retarded, compared with A . duodenale, Smith (1904) described similar upper respiratory signs and symptoms at the same time. Pulmonary or upper respiratory signs or symptoms were not observed after experimental infection of man with A . ceylanicum (Wijers and Smit, 1966). Soon afterwards, or coincident with the abatement of upper respiratory events, abdominal signs and symptoms are observed in infections of all three species. Epigastric pain has been a uniform finding (references too numerous to catalogue). The following symptoms have been described in association with abdominal pain-depressed appetite, indigestion, colicky cramping epigastric pains, a sickening ache that is more intense when the stomach is empty and is often relieved by foods and alkalis, postprandial fullness with nausea and even vomiting, and flatulence. Pain has often been described as being extremely severe and has been compared to that which accompanies peptic (Hodes and Keefer, 1945; Salem and Truelove, 1964; Bajpai and Gupta, 1966; Tandon et af., 1966) and duodenal (Yenikomshian and Shehadi, 1943: Brumpt, 1952) ulceration, and cholecystitis (Brumpt, 1958). Pain in hookworm infection has led to confusion by simulating duodenal ulceration (Ashburn, 1932; Gibbes, 1934; Leslie and Tovey, 1955) or gastric carcinoma (Matilla et al., 1951). Confusion is not surprising since radiological findings in hookworm infection and in ulceration exhibit many close affinities. Parodi (1958) suggested that right peri-umbilical pain of hookworm infection might be associated with “a paralytic ileus of Ancylostoma” and cautioned against urgent recourse to abdominal surgery in tropical regions where hookworm infection may be endemic. Brumpt (1952) stated that abdominal pain and diarrhoea, which seldom persisted for more than 15 days, were most severe after primary infection with

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A . duodenale and that symptoms of duodenitis were associated with worm maturation and start of oviposition. Much of the literature describes severe pain as common in 20-50% of chronic hookworm infections (Gibbes, 1934; Yenikomshian and Shehadi, 1943; Leslie and Tovey, 1955; Bajpai and Gupta, 1966). Pain has been described with both common species ( A . duodenale and N . americanus) and also with A . ceylanicum (Wijers and Smit, 1966; Bearup, 1967). Other primary signs and symptoms of hookworm infection, often associated with abdominal pain, are indigestion, inappetence, diarrhoea, dysentery and, less frequently, constipation. There does not appear to be any clearly defined temporal relationship between these signs and elapsed time after infection except, as might be expected, that onset of indigestion and diarrhoea are related to the arrival of fourth-stage larvae in the intestine, and dysentery is related to the development of adult worms and the start of blood sucking. Intermittent diarrhoea associated with immature adult A. duodenale is common between 2 and 6 weeks after infection (Ashford et al., 1933a; Brumpt, 1952). I n N . arnericanus infections, diarrhoea was common 7-9 weeks after infection (Smith, 1904). Dysentery was first seen 28 days postinfection with A. duodenale (Brumpt, 1958). This correlates well with findings in experimentally infected chimpanzees from which isotope-measured blood loss commenced on the 20th day, and peak losses on the 30th day were associated with the development of immature adult A . duodenale (Miller, 1968). There is little information correlating intensity of primary signs and symptoms with size or chronicity of infection. Martuscelli and Biaggi (1960) showed a significant correlation between abdominal pain and faecal egg counts in excess of 25 000 g-I, and between diarrhoea/melaena and egg counts greater than 5000 g-l. The subjects observed by Ashford et al. (1933a) had been accidentally infected at a known time but with unknown numbers of larvae; 64-1400 A . duodenale were recovered after anthelmintic treatment. The numerous signs and symptoms listed by Smith (1904, 1905), Kendrick (1934), Laederich et al. (1944), Myhre and Wallace (1956) and Brumpt (1952, 1958) were compiled from observation of experimental infections that, with A . duodenale, were often established to control polycythaemia Vera. Brumpt’s subjects were infected with 200-700 larvae and had egg counts up to 6300 g-l. Other authors described chronic infections of unspecified magnitude. B.

ANAEMIA AS A PRIMARY SIGN

Although primary signs and symptoms associated with migration of larvae and with worms in the intestine may be annoying and even debilitating, the cardinal sign of hookworm infection is anaemia. At this point, classification of signs and symptoms as primary or secondary becomes less precise. Anaemia is certainly a consequence of the worms’ blood-letting activities in the intestine, and is thus a primary sign. This is indisputable in acute canine ancylostomiasis where massive blood loss causes immediate, acute, initially

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uncompensated and often fatal anaemia. Although this type of acute haemorrhagic uncompensated anaemia does occur in the human species (Garcia Salas et al., 1971), and may have a fatal outcome (Ashford et a]., 1933a; Zimmerman, 1946; Braun, 1965), reports are extremely rare. For each report describing acute anaemia, there are more than 100 that describe chronic iron-deficiency anaemia. This is not necessarily an indication that acute hookworm anaemia is uncommon in the human species. Massive and rapidly fatal hookworm infection, particularly after primary infection of infants, may go largely unreported in the most primitive situations in backward areas, where high infant mortality is accepted as inevitable, deaths are not reported, treatment may not be available and if available may not be sought. At least one of the hookworms that infects man, A . duodenale, was shown to take sufficient blood to induce acute uncompensated fatal haemorrhagic anaemia, after experimental infection of infant chimpanzees (Miller, 1968) that were of similar weight to I-year-old children. With adequate iron reserves and in the absence of other complicating diseases or deficiencies, blood loss and initial acute uncompensated anaemia in man rapidly stimulate erythropoiesis. While iron reserves last, hookworm anaemia is normochromic and hyperplastic (Brumpt, 1958). C.

PRIMARY MORBIDITY IN CHRONIC INFECTION

Anaemia in chronic hookworm infection is more appropriately a secondary sign. Other signs and symptoms of chronic infection may be classified as primary or secondary, depending on whether their occurrence is directly associated with activities of the worms or whether they are a consequence of anaemia and hypoproteinaemia. Primary signs abate after removal of the worms, without necessarily improving-the haematological status. Secondary signs disappear after reversal of the anaemic status (e.g. after iron therapy) without necessarily first removing the hookworms. Primary signs of chronic infection are largely the same as those associated with acute infection including diarrhoea, constipation, abdominal pain, indigestion, etc. These are a direct consequence of the presence of worms since removal of all or most by anthelmintic treatment (Gibbes, 1934; Matilla et a/., 1951 ; Parodi, 1958), even without apparent haematological recovery (Yeni komshian and Shehadi, 1943), will cause their partial or complete remission. Nevertheless, in experimentally induced acute infections many primary signs and symptoms abated spontaneously (e.g. after a few months) without removal of the hookworms and the superceding chronic infection was almost asymptomatic (Laederich et al., 1944; Myhre and Wallace, 1956; Brumpt, 1958). This leads one to question whether primary signs and symptoms in chronic hookworm infection are a consequence of persistence of old worms or, more probably, caused by new worms from continuing environmental reinfection. Most chronic infections were studied in hookworm-endemic areas so that continuing reinfection was extremely likely.

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D.

SECONDARY MORBIDITY IN CHRONIC INFECTIONS

1. Anaemia Chronic iron-deficiency anaemias, abundantly described in hookworm infected people, develop gradually after a single infection (Saint-Martin and Dussault, 1957), or more commonly in the presence of continuing exposure to reinfection. Months or even years of chronic infection may pass before anaemia becomes apparent or reaches a severe level. Chronic hookworm anaemia in man is more approximately described as a secondary sign of infection since it can be prevented, alleviated or even cured by dietary administration of supplementary iron, without removal of the hookworms (Biggam and Ghalcoungui, 1934; Cruz, 1934; Rhoads et al., 1934; Cruz and de Mello, 1948; Brumpt, 1952; Foy and Nelson, 1963; Roche and Layrisse, 1966). Only in the heaviest infections or in the presence of some complicating factor or intercurrent disease does iron therapy alone fail to restore most infected subjects’ haematological values. Complicating intercurrent conditions have included unspecified dietary deficiencies (Cort, 1932), inadequate protein (Demarchi, 1958; Larizza and Ventura, 1959; Tasker, 1961), folic acid deficiency (Trowell, 1943; Daftary and Bhende, 1956; Foy and Kondi, 1958; Tasker, 1961 ; Saraya et al., 1970), interference with iron absorption by dietary chelating agents such as phosphates and phytates (Foy and Kondi, 1960) and kwashiokor (Allen and Dean, 1965; Vanier, 1966). Familial haemolytic anaemias (Trowell, 1956 ; Vanier, 1966) and chronic bacterial and protozoal infections, for instance syphilis, tuberculosis and amoebiasis (Foy and Kondi, 1960) and malaria (Allen and Dean, 1965; Vanier, 1966) also may contribute to the severity of chronic hookworm anaemia and/or the failure of iron therapy to restore haematological values. In some areas schistosomiasis is the most common complicating factor (Foy and Kondi, 1960; Farid et al., 1968; Waslien et al., 1973). Similarly, intercurrent complicating diseases that contribute to the severity of hookworm anaemia and prevent haematological recovery after iron and/or anthelmintic therapy, are aggravated by hookworm infection. Deviations of haematological findings from those typical of microcytic iron-deficiency anaemia may indicate that concurrent diseases or deficiencies are present (e.g. macrocytes and folic acid and/or vitamin B,, deficiency). Relative saturation of transferrin and presence of marrow reserves of iron are of prime importance in differential diagnosis. Chronic and, to a much lesser degree, acute anaemia are cardinal signs of hookworm infection, but if level of infection is low and/or dietary intake and reserves or iron are adequate, hookworm infection may persist for many years without evidence of clinically significant anaemia (Dick and McCarthy, 1946; Hynes et al., 1946; Walker, 1955; Bothwell and Finch, 1962). Hookworm infection is endemic in many areas of the world in which anaemia may be rare, particularly in more advanced nations. Development of irondeficiency anaemia in chronic hookworm infection is slow and insidious, but

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fortunately in uncomplicated cases it can be reversed and cure achieved very rapidly. The host-parasite relationship between man and his hookworms appears to be balanced on a knife-edge since any upset in the equilibrium (e.g. population explosion, breakdown in sanitation, natural disaster, war) can precipitate disastrous consequences. In this context de Souza (1966) described an outbreak of hookworm disease of epidemic proportions after crop failure and Zimmerman (1946) described acute fatal ancylostomiasis in children as a direct consequence of wartime occupation by belligerent military forces. Many investigators have attempted to show a correlation between worm load, usually assessed by faecal egg counts, and intensity of anaemia. All manner of relationships have been claimed, from close positive correlations, through no correlation, to even a negative relationship. The historical aspects of this relationship and parameters for a valid test have been reviewed by Roche and Layrisse (1966). The consensus is that a certain minimum level of infection is necessary to cause anaemia. This level depends on the iron-reserve status of the population, daily dietary iron intake, its availability and absorption and on hookwormmediated and other losses or iron from the body. The infection threshold for anaemia therefore varies from one population to another, within each population between different groups and families, between different ages and sexes, and from one individual to another. The kinetics of interaction between individual and group factors are discussed below. The haematophagus appetites of different hookworm species must also be considered. Variations in findings by the same author between different circumstances may be striking. Smillie (1922) noted that more than 25 N . americanus were necessary to cause anaemia, but later (Smillie and Augustine, 1926) in another population he concluded that 500 were required. Carr (1926) found no significant difference in the haemoglobin values between “normal” uninfected members of the population and those with faecal egg counts of 50-500 g-’ ( N . americanus was predominant). At the other extreme, Hill and Andrews (1942) estimated that faecal counts of less than 13 000 g-’ (approximately 500 N . americanus) were compatible with “normal” haemoglobin values in Georgia and Alabama, but in the contiguous state of Mississippi only 25 worms were required to cause anaemia. The results of Kendrick’s very large survey (1 3 000 people) in a relatively undeveloped area of southern India (1927) showed that in mixed infections of A . duodenale and N . americanus (ratio of 1/20) about 40 worms (1000 eggs g-’ of faeces) were sufficient to cause anaemia. Layrisse and Roche (1964) demonstrated sexand age-mediated differences in minimal anaemia-inducing levels of infection, equivalent to faecal counts of 2000 eggs g-I in women and children and 5000 eggs g-’ in adult males. Sex and age differences are easily explained by metabolic iron requirements, since growth, menstruation and reproduction impose additional iron stresses and increase vulnerability to “hookworm anaemia” . Only after initial minimum threshold levels of infection are achieved is it common to find that severity of anaemia is related to intensity of infection.

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Where sufficient observations were included, an inverse correlation between intensity of infection (above the population or group vulnerability threshold) and haematological values was often found to be statistically significant (Stoll and Tseng, 1925; Carr, 1926; Kendrick, 1927; Layrisse and Roche, 1964; Topley, 1968; Burman et al., 1970; Saraya et al., 1970). There is no justification to hypothesize on the significance of a particular level of hookworm infection as a cause of anaemia without prior intimate knowledge of the base-line status of each population in iron reserves, intake and absorption. To delve, therefore, in the literature into statistical protocols on the relationships between egg counts or worm burdens and haematological values is futile. There is also little value in extrapolating from and comparing observations in different surveys without detailed knowledge and analyses of the ferrokinetics underlying the observations. The degree of “hookworm anaemia” may vary from negligible (i.e. asymptomatic with respect to reduction in haematological values) to extreme. At the extreme, haelnoglobin values of 2-3% and erythrocyte counts of < lo6 mm-3 have been recorded in terminal or near-terminal cases (Ashford, 1900; Stiles, 1912; Brumpt, 1958; Somers, 1959; Borrero et al., 1961; Bajpai and Gupta, 1966; Fowler et al., 1968). It is surprising that depressions of haematological measures close to these terminal values may be compatible with continuing physical activity by many infected individuals. Where chronic anaemia is slowly progressive, physiological compensation by the circulatory system may permit active participation in exercise and even continuing work by severely anaemic subjects having haemoglobin values as low as 4 % (Foy and Kondi, 1957) and erythrocyte counts of lo6 mmP3 (Brumpt, 1958). Such people are of course existing in a most hazardous situation since the threat of cardiac insufficiency and congestive cardiac failure are always present. Severe and potentially fatal circulatory failure is common in hookworm-infected subjects with haemoglobin values of less than 5 % (Hill and Andrews, 1942; Janssens, 1955; Somers, 1959; Fowler et al., 1968). Sudden death may be associated with stress factors such as second-stage labour (Wickramasuriya, 1937) and overload of a hyperkinetic circulation after intravenous blood transfusions (Fowler et a]., 1968). Reduction in circulating plasma volume after use of diuretics to cure hypoalbuminaemic oedema in severe chronic hookworm infection may also prove catastrophic.

2. Hypoalbuminaemia and Oedema References to hypoproteinaemia and hypoalbuminaemia in chronic hookworm infection, although less frequent than anaemia, are too numerous to catalogue in detail. Significant correlations have been shown between the intensities of each of the following parameters : hookworm infection, anaemia, hypsproteinaemia and hypoalbuminaemia (Saraya et al., 1970). Hypoalbuminaemia and oedema appear to develop in persistent chronic infections after haematological values have reached grave or near-terminal levels. Haemoglobin values of 2.56 g 100 ml-l (Fowler et al., 1968) and less than 2 g 100 ml-I. (Farid and Miale, 1962) were correlated with oedema.

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The aetiology of oedema is not primarily anaemia but is a direct consequence of the hypoproteinaemia and hypoalbuminaemia of chronic severe hookworm infections. Plasma protein less than 5 g 100 ml-l with plasma albumin less than 3 g 100 ml-* (Villela and Teixeira, 1937; Charmot and Reynaud, 1962; Gilles et al., 1964; Bajpai and Gupta, 1966) and reversal of the A/G ratio (Brumpt and Sang, 1955) were shown to be related to oedema. Martuscelli and Biaggi (1960) showed a statistically significant correlation between oedema and egg counts of more than 5000 g-’ and explained oedema through interference with protein absorption, a questionable assumption. Oedema in chronic hookworm infection has been recorded subcutaneously in dependent limbs (MacGregor, 1944), and extensively as generalized anasarca with pericardial and pleural effusion and ascites (Ashford and King, 1907; Roche et a]., 1957; Charmot and Reynaud, 1962; Gilles et al., 1964). Oedema is uncommon at younger ages, presumably because of insufficient and inextensive infection. Only one report of oedema in infants was found (Juminier and Zakine, 1960). In a hookworm-endemic area, famine and failure of the prime protein crop increased the incidence of hookwormassociated oedema, anasarca and ascites to epidemic proportions (de Souza, 1966). 3. Other Sequelae Numerous other secondary signs and symptoms, attributable to anaemia and hypoproteinaemia, accompany chronic hookworm infection. The most important of these are associated with progressive cardiac insufficiency and congestive failure. References are numerous and the list includes weakness, breathlessness, palpitation, tachycardia, cardiac dilation, venous distention, low blood pressure (rarely), systolic Murmurs, angina pectoris, vertigo and fainting. Congestive cardiac failure was shown to be reversible after correction of anaemia (Darke, 1959), when the oedema and other circulatory signs were also alleviated. Electrocardiograms of profoundly anaemic, chronic hookworm cases have been described as abnormal (Calo, 1957; Shaper and Shaper, 1958; Borrero et a/., 1961), particularly that portion associated with the repolarization phase (Perroni and Pancaldo, 1953; Nhonoli and Chukwuemcka, 1971). Calo (1957) proposed a toxic aetiology but this was dismissed by Perroni and Pancaldo (1953) who stated that abnormal e.c.g.’s were associated with haematological values of 2-3.5 lo6 erythrocytes mm-3, although anthelmintic treatment produced a normal e.c.g. before haematological values were fully restored. Shaper and Shaper (1958) described considerable but not complete normalization in e.c.g. changes (low voltage and flattened “T” waves) as haematological values returned to normal after treatment of severe chronic anaemias attributed to hookworm infection. However, others (Hill and Andrews, 1942) failed to observe any e.c.g. changes even in severe cases. Cardiac dilation is a common clinical finding (MacGregor, 1944; Shaper and Shaper, 1958; Borrero et al., 1961; Charmot and Reynaud, 1962), confirmed at necropsy of subjects with overwhelming infections (Zimmerman,

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1946). The heart shadow was shown to double in all dimensions (Hill and Andrews, 1942). Reduction in anaemia was accompanied by reduction in size (Shaper and Shaper, 1958). Changes in the circulatory system and associated signs and symptoms may be attributed almost entirely to anaemia and physiological compensation, for instance by increase in blood volume, to establish a hyperkinetic circulation (Beet, 1956; Somers, 1959; Fowler et a/., 1968; Areekul, 1974). Although there are conflicting opinions on increased total blood volume, there is a consensus supporting increase in the plasma component (Boycott, 1911; Borrero et a/., 1961; JaIili and Hindawi, 1962; Fowler et a/., 1968; Areekul, 1974). Cardiac dilation and increased rate and stroke volume also contribute to establishment of a hyperkinetic circulation (Beet, 1956), when the haemoglobin value falls below 60% (Razzak, 1965). Shortening of the pulmonary circulation time (determined by isotope distribution) was reported. Maintenance of circulating blood volume may also be effected by vasoconstriction, as proposed by Saif (1968), who failed to detect increase in blood volume, except in uncompensated cases with oedema. Prolonged chronic anaemia from hookworm infection induces other more subtle changes in the health of an individual or population. As well as inability to work, and therefore to contribute adequately to the maintenance and well-being of individuals and dependents, mental processes and aptitude may be impaired leading to dullness, depression, confusion, poor memory and an appearance of stupidity (Arora, 1951; Borrero et al., 1961). Stiles (19 12) reported that in chronic hookworm infection childrens’ physical and mental development were stunted so that 21-year-olds might only be equivalent to 14-18-year-old uninfected subjects. Children with chronic hookworm infections appeared to be the most stupid in their class. In a subsequent report, Stiles (1932) expanded this theme and stated that the most backward children were chronic hookworm cases and marked improvement in both physical and mental performance could be stimulated by anthelmintic therapy. These observations years ago in a developed country prompts one to question how much unnecessary waste of educational potential occurs today in developing areas in which hookworm infection and chronic anaemia are endemic. A wide range of reproductive disorders have been attributed to the effects of hookworm infection, with or without anaemia (Sandwith, 1894; StiIes, 1912; Larrieu, 1957). Impaired physical development in children included delayed puberty. Chronic hookworm anaemia and congestive cardiac failure may be disastrous during parturition. Wickramasuriya (1937) attributed about one-quarter of maternal mortality and over 70% of foetal (miscarriages, abortions, stillbirths), neonatal and early infant mortality to hookworm infection and anaemia and stated “there is not any greater menace to the expectant mother and her unborn child than hookworm disease”. He alleged hookworm anaemia was a greater cause of abortion and stillbirths than congenital syphilis. Complications of pregnancy (toxaemia, pre-eclampsia and eclampsia) were also many times more common in anaemic patients. De Azevedo (1965) attributed delayed puberty, abortions,

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decreased birth rate, etc., to hookworm infection but did not incriminate anaemia as the intermediate cause. He described these conditions as common in hookworm-infected individuals who were not also anaemic and maintained that the presence of worms and the “toxic” action were responsible. Evidence for a primary toxic aetiology in hookworm infection, to the partial exclusion of chronic anaemia, is not convincing. Findings on loss of weight are most inconsistent, and are clouded by nutritional influences. Severe hookworm infection does not necessarily cause weight loss, particularly where the course is acute and diet adequate (Ashford, 1900; Chandler, 1929; Roche and Layrisse, 1966), although some describe severe loss of weight in acute infections (Ashford et al., 1933b; Hodes and Keefer, 19453. In chronic hookworm infections maintenance of body weight depends on diet, severity and chronicity; weight loss has been shown to be common (Stiles, 1912; Smillie and Augustine, 1926; Roche et al., 1957). Statistical correlations between loss of weight and the intensity of infection have been described (Crowley et nl., 1956). Many additional secondary signs and symptoms attributed to hookworm infection may be attributed to other conditions; including splenomegaly and hypersplenism in malaria, macrocytic anaemia in folic acid deficiency, several biochemical aspects of digestive dysfunctions in tropical sprue and parotid enlargement in primary protein malnutrition. Even after discarding these salient exceptions, there still remains a long list of signs and symptoms that have been described in subjects with chronic anaemia and hookworm infection, some frequently, others rarely. The aetiology of these changes and their relationship to hookworms and/or chronic hookworm anaemia are not always apparent. References are distributed widely, and repetitively, throughout the literature. Included in the list are: depigmentation of skin and hair in Negroes, koilonychia, glossitis, stomatitis, pyorrhoea, papilliform skin eruptions. hypothyroidism, renal and hepatic dysfunction, pellagra and polyneuritic signs, achlorhydria, joint pains, retinal haemorrhages and headache. Abnormal behaviours have been observed, including pica and geophagia. Iron loss in chronic hookworm anaemia and depletion of tissue iron appear likely to be the aetiological agent in several of these, for instance depigmentation, glossitis, achlorhydria, koilonychia, chelosis and other epithelial disorders, pica and geophagia. Other signs have been attributed to the postulated toxic effect of the parasite, but attempts to show toxins seem to have depended on hypersensitive reactions to preparations of larvae or adult worms. Such hypersensitive or “toxic” reactions are as likely to be mediated by the bacterial flora associated with the larvae and worms used to prepare antigens, as they are to be associated with proper nematode proteins and antigens. For instance, Ashford et al. (1933a) suggested that skin reactions and generalized pruritis (without urticaria) were mediated by hypersensitivity after the death of aberrant hookworm larvae with subsequent liberation of allergens. These reactions perhaps might also be stimulated by bacteria to which the subject was also sensitive. Whether hookworms elaborate toxic products (excluding allergens) remains open.

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INCIDENCE OF MORBIDITY

It is often impossible to detect a relationship between figures for incidence of infection and incidence of morbidity. This relationship and variations thereof have been exhaustively discussed by Roche and Layrisse (1966). Shaper and Shaper (1958) noted that anaemia due to hookworm infection was the second most common cause of admission to the medical wards at Mulago Hospital, Kampala, and that hookworm infection was the commonest cause of anaemia and was often associated with circulatory failure, with oedema and hypoproteinaemia. This situation prevailed, essentially unaltered, 13 years later (Pate1 and Lwanga, 1971). They also stated that admission rates for this syndrome were highest immediately after the rain season. In Mauritius, anaemia associated with hookworm infection ranked as the second most common cause of hospitalization (Anon, 1964). Second to schistosomiasis, hookworm was classified as the most important parasitic disease in Egypt (Aly et al., 1962). Ashford (1900) described hookworm anaemia as the most destructive disease in Puerto Rico. Although economic development has changed this situation in Puerto Rico and other countries that have developed, there are still many backward areas in the world in which similar conditions prevail today. Trowell (1960) reviewed anaemia as a common cause of hospitalization and death, principally in tropical Africa, with the following figures for heart disease attributable to anaemia, Rhodesia 5.8%, Uganda 2.4%, Nigeria 9.5%, Jamaica 0.8%; and for deaths from heart disease attributable to anaemia, Ghana 0.5%, Uganda 5.2%. Unfortunately all references could not be pursued to source to determine what proportion of anaemia was iron-deficiency or to assess the aetiological significance of hookworm infection. Where the original reference was available, it was found that iron-deficiency anaemia associated with hookworm infection comprised a major proportion of the statistics. Variations in morbidity in hookworm-endemic areas may be attributable to size of worm burdens and diet, particularly to iron absorption. With an iron-rich diet, high intensities of hookworm infection (e.g. egg counts of 5000 g-l) may not cause morbidity (Fulleborn, 1929). With inadequate diet, low worm burdens have been described as being associated with significant morbidity. This latter is the more common situation in some developing countries in which hookworm infection with anaemia is endemic. Two other factors have been considered to influence the relationship between morbidity and infection. One, race or breed of the host, has been occasionally proposed but there is little valid statistical evidence (Smillie and Augustine, 1925) that negroes may be less susceptible to infection and to the effects of infection than Caucasians. In the comparative fields, however, there is now valid evidence of breed variations in resistance to morbidity. Whitlock and Georgi (1968) showed that sheep with certain genetic and phenotypic characteristics were more susceptible to severe morbidity and mortality with low-grade infections of Haemonchus contortus. The second potential moderating influence, immunity, is an aspect of the man-hookworm relationship about which we still know very little. In many

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comparative systems immunity is a most important factor in moderating pathogenesis and reducing morbidity. If, as seems likely, acquired immunity in hookworm infection of man has significant protective effect in the manhookworm relationship, then it could be expected, by analogy with the dog-hookworm system (Miller, 1971) to act through two mechanisms. Acquired resistance to reinfection would limit first, the rate and success of establishment of new hookworms and secondly, the blood sucking and pathogenic mechanisms of the few worms that managed to evade the immunological defenses would be impaired. Adults therefore would carry smaller burdens and these worms would be less pathogenic than in children. Unfortunately, there is insufficient data in the literature to support or negate such hypotheses.

VI. MORTALITY The literature on hookworm mortality in man is relatively rare and most relates to conditions prevailing 50 years ago. There is no doubt, however, that infection with human hookworms, especially A . duodenale in infants and young children, can cause severe acute rapidly fatal enterorrhagic disease analogous to A . caninurn in dogs (Miller, 1971). Charmot and Reynaud (1962) reported a 7 % death rate in children soon after admission to hospital and earlier, Ashford and King (1907) attributed 25 % of deaths in Puerto Wico to hookworm. Deaths were common both in children and in adults (Ashford, 1900) and were observed as early as 30 days after massive primary infection. Schapiro and Nauck (1931) attributed 71 % of child mortality and 42% of all mortality in a charity hospital to hookworm anaemia. More recently Bwimbo (1970) reported hookworm anaemia as. the third most frequent cause of death in children 1-5 years old in Uganda, being responsible for 4 and 7 % mortality in children of 1 month to 1 year and 1-5 years old respectively. Death from cardiovascular inadequacy and circulatory collapse as a terminal effect in chronic hookworm disease, as distinct from acute enterorrhagic mortality, was also relatively frequent (Trowel), 1956; Fowler et a]., 1968) although it is difficult to differentiate the relative contribution or importance of other intercurrent diseases in this group. Although deaths from hookworm infection may have almost disappeared in some areas (Yokagawa, 1967), and 50-75 years ago environmental and economic conditions, medical services and level of development were certainly lower in these areas (Puerto Rico, Costa Rica, Central Africa), there are still areas in the world where similar conditions prevail. This is especially true of areas of international and civil belligerency and in their refugee populations (Thomas et al., 1971). In this respect, Zimmerman (1946) reported a 50 % death rate from acute hookworm enterorrhagia ina population of children in an area where many of the attributes of civilization were suspended after occupation by belligerent military forces. It would be interesting, and no doubt depressing, t o review the medical consequences related to hookworm and death today and in the future in areas of the world in which development and civilization are, or may be, in similar jeopardy.

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In backward primitive conditions that still prevail in many areas of this “civilization” high infant and child mortality is accepted as normal, medical and reporting sciences are unavailable, or if available are not used for acute conditions in children. In the past and even today the incidence of acute hookworm mortality may be higher than believed or reported.

VI I. PATHOPHYSIOLOGY The primary pathophysiological activity in hookworm infection of man, is, without doubt, enterorrhagia and iron loss. Blood is sucked and passed directly through the worms (Roche and Martinez-Torres, 1960), probably for dietary and respiratory functions, is spilled around their heads in the feeding area and may also leak from damaged and from thrombosed mucosal blood vessels in old feeding areas (Kalkofen, 1970). Much contradictory literature on hookworm pathophysiology has accumulated over the past 40-50 years but this represents one of the few areas of the man-hookworm relationship that is now clear. Roche and Layrisse (1966) reviewed the data in this area of hookworm pathogenesis most adequately: only later information will be discussed here. A.

BLOOD LOSS

The most widely accepted modern methods of determining blood loss are by labelling circulating erythrocytes with a radioactive isotope, ideally W r as sodium chromate. Faecal clearance of the slCr (not reabsorbed from the gut) is calculated as whole blood from the relative radioactivities of the total daily faecal output and of peripheral whole blood. Values are expressed as ml of blood per worm per day, discounted usually for control or post vermifuge faecal radioactivity losses. Although this technique has several potential but minor inaccuracies, it has been the yardstick and has provided comparable results from different workers. This field has also been well reviewed by Roche and Layrisse (1966) and subsequent reports (Mahmood, 1966; Martinez-Torres et a/., 1967; Diez-Ewald and Layrisse, 1968; Megahed et al., 1972a) have not provided contradictory or significantly different results. The results clearly support the established contention that A . duodenale has a much larger appetite for blood than N . americanus in an approximate ratio of 10 : 1. Values from mean daily blood loss due to N . americanus range from 0.01 to 0.04 ml per worm, while for A . duodenale the range is from 0.05 to 0.3 ml per worm. Although some have measured blood loss by Iabelling circulating erythrocytes with 59Fe,this label tends to provide lower values since a variable and unpredictable amount of the iron is reabsorbed by the gut. Measurement of blood loss with W r with whole body counters have been used in studies of erythrocyte survival and blood loss in man (Giordani et al., 1967; Holt et al., 1967) and in blood loss in canine hookworm infection, but not yet in blood loss and erythrocyte survival studies in hookworm infection of man. Evidence of relationships between blood loss and other measures of infection and morbidity have been sought. Statistically significant correlations

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between blood loss and faecal egg counts were demonstrated by Arakawa (1961), de Azevedo et a/. (1965a), Farid et a/. (1965a) and Martinez-Torres et al. (1967). Values were 2-14 ml ( f 1.01 ml) per thousand eggs per gram of faeces per day for N . americanus (Martinez-Torres et al., 1967), and 4.47 ml (k 1.16 ml) for A. duodenale (Farid et al., 1965a). Rate of blood loss was claimed to be proportional also to the number of worms (Farid et a/., 1966; Areekul et al., 1970b) or inversely proportionaI to the logarithm of worm numbers (Tasker, 1961). Mahmood (1966), however, found relationships between blood loss and worm burden not to be significant. Some of the apparent contradictions may be explained by observations by Tasker (196 I), de Azevedo (1965) and Areekul et al. (1970b) that daily blood loss per worm varied with intensity of infection, being less in heavier infections. Tasker (1961) calculated 0.03 ml and 0.1 ml for heavy and light infections respectively. De Azevedo et al. (1965a) reported 0.0024006 ml in infections of more than 300 N . americanus and 0.01-0.03 ml when less than 150 worms were present. This phenomenon was also observed in the dog-A. caninum system (Devakul et al., 1966; Miller, 1966c, 1968). Roche et al. (1957) noted that daily blood loss per worm appeared larger in more anaemic subjects and decreased as haematological values improved following iron therapy or blood transfusion. This finding is at present inexplicable, except perhaps by an hypothesis that anaemic blood is an inferior diet for the worms. Data from isotopic studies of blood loss in human hookworm infection have been accumulated from people with natural infections, in which age of worm was unknown. This may represent a stable situation in that only mature worms are present although reinfection may be continuing so that a dynamic situation exists in which some worms are relatively young and others are approaching senility. Most comparative blood loss studies have been conducted in experimentally infected animals in which there exists a dynamic situation mediated by rapid worm growth and development. In such a dynamic situation blood loss from chimpanzees after experimental infection with A . duodenale was shown to vary with age of worm. Owing to the extreme lethality of this infection, the period during which blood loss was observed was short compared with the potential life span of this hookworm and did not include a steady state with mature or old worms. From the results of these studies in chimpanzees and studies with A. caninum in dogs, it is clearly established that blood loss commences with the 4th moult and development of the fifth stage or the immature adult hookworm. In A. duodenale this moult occurs about 21 days after infection (Miller, 1968). If the same applies to other human hookworms then patterns of blood loss may be derived from knowledge of the endogenous development parasitic of N . americanus and A . ceylanicum. Blood loss in N . americanus infection would therefore commence during the 4th week after infection and in A . ceylanicum at the end of the 1st week. By analogy with findings of a rapidly escalating blood loss associated with the maturation and early reproductive activities of A. duodenale and A . caninum in chimpanzee and dogs, it is not surprising that sample investigations of blood loss in man in hookworm-endemic areas should give such variable and apparently irreconcilable results. In endemic M

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conditions, considering superimposed physiological effects on human morbidity of iron reserves and dietary intake, iron losses from other causes and variable worm age, it is not surprising that there was little or no evidence of a relationship between intensity of infection, blood loss and occurrence or degree of anaemia (Aly et al., 1962). In the dog-A. caninum system (Miller, 1967b, 1968) immunity inhibits or reduces blood loss. In the man-hookworm system, if immunity is an effective factor, it is anticipated that its influence would be exhibited by a similar protective effect in reducing blood loss per worm. This also may be responsible for some wide variations in figures on blood loss, for instance the disparity between figures for daily blood loss of 35 ml from patients in Kenya with 1500 N . americanus (Foy et al., 1958) and the corresponding finding in Venezuela of 250 ml from infections of similar magnitude with the same hookworm (Roche et al., 1957). The ratio between these two figures are consistent with the canine hookworm results. Indeed, there are so many potential moderating factors that the uniformity of some results is almost surprising and probably fortuitous. Little has been said about the third hookworm of man, A. ceylanicum, except in the area of primary signs and symptoms after experimental infection of volunteers (Wijers and Smit, 1966). Isotope studies in man have not been reported. However, this hookworm develops successfully in experimental hosts (i.e. dogs and cats) in which isotopic studies of blood loss are relatively easy to conduct. Rep (1966b) and his co-workers (1971), using SICr-labelled erythrocytes in dogs harbouring in excess of 10 000 worms, showed that during the dynamic evolving phase of infection (including the prepatent period) considerable losses were occurring. The figures of Rep et al. (1971) for postpatent losses from dogs experimentally infected with A. ceylanicum were equivalent to only 1 % and 10% of the daily figures (per worm) from infection in man with the two primary species ( A . duodenale and N . americanus respectively). It would therefore require burdens of extraordinary magnitude (e.g. in excess of 10000) for A . ceylanicum to cause significant blood loss and anaemia in man. In view of the sporadic and rare occurrence of A . ceylanicum in man, it is debatable whether blood loss associated with this hookworm has any significance. B.

REDUCED LIFE SPAN OF CIRCULATING ERYTHROCYTES

Earlier literature refers to haemolytic processes in infected subjects and to haemolysins in hookworms or in their extracts. The strong emphasis on iron-deficiency anaemia led to haemolytic theories being discarded, until recently. Renewed interest in haemolysis as a pathogenic mechanism in hookworm anaemia was stimulated by finding shortened survival of W r labelled erythrocytes, after correction for faecal isotope losses, in infected and anaemic subjects (Saif, 1959; Roche et a]., 1960; de Azevedo et al., 1965b; Layrisse et al., 1965; Farid et al., 1965a,b, 1966; Roche and Layrisse, 1966; Diez-Ewald and Layrisse, 1968; Megahed et al., 1972b). However, other authors (Gilles et al., 1964), or the same authors on other occasions

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(Roche et al., 1960), failed t o detect any decrease in half-life of SICr-labelled erythrocytes in severe iron-deficiency anaemia associated with hookworm infection. Moreover, when reduced half-life was noted, there were no correlations between reduction in half-life and amount of isotope, measured as whole blood equivalent, lost in the faeces (Farid e t a ] . , 1965a; Layrisse et a / . , 1965). Inherent inaccuracies in this method of estimating half-life are considerable (McCurdy, 1969), and can be circumvented by the use of a superior label, 32D.F.P. (diisopropylfluorosphosphate). Using 51Cr and 32D.F.P. labels simultaneously, Layrisse et al. (1965) and Diez-Ewald and Layrisse (1968) confirmed that the half-life of erythrocytes in hookworm-infected anaemic subjects was indeed reduced and demonstrated that this reduction had two constituent parts. The first was associated with uncorrected loss of isotope in faeces as haemorrhage and the second resulted from an increased rate of destruction of erythrocytes in the spleen (Megahed et a / . , 1972~).The first constituent was neutralized by removal of the worms and the abnormal rate of erythrocyte destruction regressed on recovery of haemoglobin values after administration of iron, irrespective of whether the worms were removed by anthelmintic treatment or were permitted to remain (Martinez-Torres et a / . , 1967; Diez-Ewald and Layrisse, 1968). Reduction in apparent half-life of the circulating erythrocytes, without hookworm infection, has been shown to be common in iron-deficiency anaemia (Rash et a / . , 1958; Verloop et a / . , 1960; Layrisse et a / . , 1965; Diez-Ewald and Layrisse, 1968), although not universal (Temperley and Sharp, 1962; Prichard, 1966). In other species results of studies of irondeficiency anaemia support the hypothesis that reduced life span, which may or may not be detectable with the 5'Cr label (Huser et al., 1967; McKee et a / . , 1968; Card and Weintraub, 1971), is a consequence of intrinsic defects in erythrocytes formed in these circumstances. The severity of the apparent reduction in half-life of circulating erythrocytes was shown (Huser et al., 1967) to be related to chronicity of the anaemia rather than to its immediate severity. Reduction in apparent half-life of SICr-labellederythrocytes also occurred in protein malnutrition accompanied by anaemia (e.g. kwashiorkor or marasmus), and was apparently associated with protein, rather than iron deficiency, as the half-life returned to normal after protein supplementation without iron therapy and in continuing anaemia (Lanzkowsky et al., 1967). The most critical factor in hookworm anaemia is iron deficiency; therefore increased rate of erythrocyte destruction and reduced half-life may have little practical significance in view of the availability for continuing erythropoiesis of iron from haemolysed erythrocytes. Other evidence concerning haemolysis and the site thereof (i.e. whether intravascular or by increased sequestration in the spleen) as indicated by biochemical findings in bile pigment metabolism, haemoglobinaemia, haptoglobulins, haemoglobin turnover and erythrocyte fragility, have been discussed adequately elsewhere (Roche and Layrisse, 1966).

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IRON BALANCE

It has been established that hypochromic anaemia is the most important factor in the morbidity and mortality of chronic hookworm disease, that blood loss as enterorrhagia is the prime factor causing anaemia, and that iron therapy may be sufficient to support haematological recovery without necessarily removing the worms. It is necessary therefore to consider all factors associated with balancing the iron account in hookworm-infected subjects. A rapid assessment of the state of iron balance can be achieved by three measurements, serum iron, iron-binding capacity of serum transferrin and the presence of haemosiderin. The literature on iron-binding capacity, serum and tissue iron has been adequately reviewed by Roche and Layrisse (1966). On the income side is total dietary intake of iron. Efficiency of iron absorption varies with different food and availability of the iron, and may be further modified by other dietary factors. For instance, iron in vegetables is poorly absorbed compared with iron in high-protein diets and in food of animal origin (Layrisse et al., 1969). Dietary iron supplementation and utilization of non-heme iron is also rather ineffective in subjects with diets deficient in animal protein (Layrisse et al., 1973, 1974). Large quantities of chelating agents in the diet, such as phytates and phosphates, interfere with absorption of available iron. Iron absorption is, however, enhanced in iron-deficient subjects. Hypochlorhydria and achlorhydria have been described in iron-deficiency anaemias. Whether reduced gastric acidity precedes, or is a consequence of, iron-deficiency anaemia is not clearly established. Adequate gastric function with low pH assists in maintaining solubility of inorganic iron and facilitates efficient absorption of dietary iron. However, there is no evidence on whether achlorhydria associated with hookworm infection interferes with iron absorption since absorption of supplementary iron in simple chemical combinations is not significantly impaired in subjects infected with hookworms, whether anaemic or not. Dietary iron supplementation is sufficient to compensate for, and to cure the anaemia associated with hookworm infection. Unfortunately, in areas of the world in which hookworm is endemic and in which iron deficiency is common, dietary sources of iron are those in which iron content is low and from which iron is most inefficiently absorbed. These individuals often barely maintain iron balance in ordinary circumstances without having to compensate for the extraordinary iron losses of hookworm infection. Under normal conditions iron is conserved by the body with little being lost, other than minimal amounts in normal sloughing of superficial cells of skin and digestive tract. These losses can usually be compensated by iron even from some poorer quality foods. Iron loss in sweat, like cellular losses, is relatively unimportant. Additional iron is required by the growing subject. Men are therefore unlikely to suffer from iron deficiency. The female reproductive cycle (i.e. menstruation, pregnancy, parturition and lactation) represent a most important loss of iron. Growing children and women in the

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reproductive years comprise especially vulnerable groups. A more critical situation exists in young females who are both growing and experiencing the additional burden of iron loss with the menarche. In certain countries or tribal groups child or early marriages are encouraged or permitted so that continuing growth requirements for iron may be compounded by the whole spectrum of reproductive requirements including pregnancy, parturition and lactation (Foy and Kondi, 1957). Therefore under good conditions in the underprivileged areas of the world iron requirements of these vulnerable population groups are often not satisfied. When combined with pathological iron loss in hookworm infection these subjects face an almost impossible task in meeting their iron commitments and in maintaining solvency from dietary income (i.e. intake modified by absorbability). Even in developed countries under good conditions of diet and health, women of reproductive age often have difficulty in maintaining iron balance. That whole blood is lost into the intestine in hookworm infection is no longer in doubt. The approximate amount has been assessed by isotopic labelling experiments with W r and can be considerable. The measurement of iron loss in faeces by isotopic labelling with 59Feprovides another pathophysiological parameter. Simultaneous double labelling with W r and 59Feto measure blood and iron loss respectively, can be used to measure the amount of haemoglobin iron (12-80 %) readsorbed from the enterorrhagia of hookworm infection (Roche and Perez-Gimenez, 1959; Foy and Kondi, 1960; Layrisse et al., 1961; Areekul et al., 1970b). Haemoglobin iron is reabsorbed, not as ionized iron but as an iron-porphyrin complex, by a route different from that by which ionized iron is absorbed (Callender et al., 1957; Turnbull et al., 1962; Brown et al., 1966; Conrad et al., 1966). Faecal iron loss in hookworm infection is therefore less than might be anticipated from blood loss figures. Estimates of iron loss in hookworm infection, based on W r derived figures for apparent erythrocyte loss (Farid et a[., 1965a), are probably of little value (Farid et al., 1970). Several publications (Layrisse et al., 1961 ; Roche and Layrisse, 1966) include hypothetical calculations that convert these observations into exemplary dynamic equations of iron balance and kinetics. Since there is no new information, the reader is referred to the original articles. The following points are implicit or explicit in the equations and calculations and are worth summarizing. (i) Hookworm infection and intestinal blood loss do not necessarily result in anaemia in subjects with normal iron reserves, if the daily iron loss does not exceed the amount adsorbed from the diet (i.e. net losses are zero). (ii) Even if iron losses exceed daily iron intake, it requires some time for net losses to exhaust iron reserves, depending on the size of iron reserves and on number and species of hookworms. If iron reserves are low at first infection (e.g. in children in underprivileged groups, or in multiparous women) anaemia may develop soon after the hookworms of a primary infection mature. If, however, hookworm infection results in a low net iron loss from subjects with replete iron

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reserves (e.g. adult males) several years may elapse before these reserves are exhausted and anaemia develops. (iii) When iron reserves have been depleted, iron losses are taken from the remaining available iron represented by the circulating haemoglobin. Chronic anaemia usually develops slowly and haematological values continue to decline until daily net losses of iron, reduced by lower haemoglobin values of the anaemic blood, become zero. This example assumes that worms cause the same losses of anaemic as of normal blood. A steady state then prevails in which there is no further reduction in haematological values since iron losses from enterorrhagia with anaemic blood are balanced by dietary adsorption of iron. (iv) These hypothetical calculations depend on constant levels of hookworm infection, constant worm haematophagus appetites and stable diet of the subject. After reaching the hypothetical balanced state of anaemia, exhausted iron reserves and continuing blood loss with zero net iron loss, any superinfection of hookworm with an increase in blood loss unbalances this equilibrium and haematological values proceed to decline until a new equilibrium with zero net iron loss is established with more severe anaemia. (v) Any change in worm burdens results in a change in haematological values and iron status. Rhythmical patterns of blood loss associated with intrinsic worm physiology, loss of senile worms or anthelmintic treatment result in improvement in iron status, depending on diet. Net gain of iron is reflected in improved haematological values. The physiological conservative emphasis is initially on improvement of haematological values. These return to normal before significant iron reserves are accumulated. However, since small amounts of iron may be available or are absorbed from the diet, recovery of normal haematological status from severe anaemia may require months or years. (vi) Therapeutic administration of iron, whether orally or parenterally, immediately satisfies significant portions of the iron deficit and results in rapid recovery of normal haematological values. (vii) The potential importance of acquired immunity must also be considered since “self-cure” reactions (i.e. worm expulsion similar to those seen in some comparative host-parasite systems) would produce a favourable unbalance of the equilibrium and haematological recovery. Similarly, immunological reduction of worm haematophagus potential, as observed in the dog-A. caninum system, would result in slower decline of iron reserves or haematological values, equilibrium, or a slow recovery of haematological values even in the presence of otherwise significant persistent worm burdens. These summarized hypotheses and scenarios are based on observed fact and are essential to the comprehension of the kinetics of chronic irondeficiency anaemia of hookworm infection.

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Acute anaemia, analogous to the experimental canine system ( A . caninunt) observed after large primary infections of infants and children, does not fit within this system but exhibits the temporary initial lag phase in effective mobilization for increased erythropoiesis. Depending on the iron reserves and net losses, this situation either develops rapidly into chronic anaemia, which fits the hypothetical system, or progresses to an earlier fatal termination. Epidemiological observations on incidence of morbidity and mortality in hookworm infection and of anaemia in underprivileged areas of the world illustrate these interacting factors and provide corroborative evidence of the prime importance of iron intake, availability and absorption. D.

PLASMA PROTEIN LOSS

Intestinal haemorrhage and loss of whole blood and iron have been adequately documented. Measurements using radioisotopes ( W r as sodium chromate, 32Pas phosphate, 59Feas ferric chloride) measure strictly the rate of clearance of erythrocytes from circulation to intestine or loss of haemoglobin iron from the body (59Fe). These techniques do not provide exact measurements of the amount of plasma that accompanies labelled erythrocytes to the lumen of the intestine. It is assumed that the ratio of erythrocytes to plasma in the intestinal haemorrhage is the same as that of peripheral venous circulation from which periodic reference blood is withdrawn. There are many factors that can influence this relationship and this assumption may or may not be valid. Factors include potential variations of packed cell volume induced by the methods of obtaining peripheral venous blood and differences between packed cell volume of peripheral venous blood and of blood in the capillaries in the submucosa of the intestine. Fasting state or digestive function has considerable influence on the latter. Even if one accepts that approximations estimating whole blood loss from erythrocyte clearances are potentially questionable, the techniques nevertheless have served useful functions in elucidating the basic pathophysiology of hookworm infection. More accurate methods of assessing loss of plasma to the intestine are available. Detection of plasma leakage and measurement of exogenous catabolic rates of plasma albumin and total exchangeable albumin pool have been accomplished by isotopic tracer techniques in which albumin or plasma is labelled with lZ5I,l3IT, 5'Cr (as chromic chloride) or 95Nb.Plasma leakage can also be assessed by measuring faecal clearances of synthetic polymers (PVP) or macromolecules (iron-dextran) labelled with I3'I or 59Fewith similar physical-biological characteristics to plasma proteins, except for their degradability (Jarnum, 1963; McFarlane, 1964; Van Tongeren and Majoor, 1966; Dargie et al., 1968). It is necessary to differentiate between normal plasma exudation t o the intestine, which constitutes the pathway for exogenous catabolism of plasma albumin, and superimposed pathological leaks. In a number of gastrointestinal helminthiases abnormal plasma leakage to the intestine contributes to excessive exogenous catabolic rates of plasma albumin and hypoalbuininaemia without concomitant intraluminal haemorrhage or loss of erythrocytes. This pathophysiological mechanism has been

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postulated or described in a variety of host-parasite relationships, e.g. Nippostrongylusbrasiliensis infection in rats (Urquhart et al., 1965), Ostertagia ostertagi in calves (Halliday et al., 1968), Strongyloides ransomi in pigs (Giese et al., 1973), Ostertagiu ostertagi in calves (Halliday et a/., 1968), U. stenocephala and A . braziliense in dogs (Miller, 1971). These pathological plasma losses do not appear to be a consequence of, or associated with, feeding activities of worms but have been shown to result from inflammation including an increased capillary permeability (Urquhart et al., 1965; Barth et al., 1966) accompanied by ultramicroscopic changes at the intercellular level (Murray, 1969; Murray et al., 1970a,b). It is very probable that the same processes occur in hookworm infection of man, superimposed on haemorrhagic blood loss, although this has not yet been fully described or proven. Measurements of plasma protein loss to the intestine of anaemic hookworm-infected human subjects have been made by Gilles et a/. (1964) and Blackman et al. (1965), who described reductions in total exchangeable albumin pool and hypoalbuminaemia. Gilles et al. (1964), measuring plasma loss by labelling circulating albumin with 1311, showed that albumin loss exceeded values that would be anticipated from whole blood loss measured by Wr-labelled erythrocyte clearances. Blackman et al. (1965) reported albumin losses from 17 subjects that indicated an excessive rate of exogenous catabolism over controls. The magnitudes of the losses were significantly related to numbers of worms; the mean value approximating 0.1 g of albumin or 3 ml of plasma per 100 N. arrrericanus. When corrected for packed cell volumes of the anaemic subjects, the mean value was equivalent to a daily loss of whole blood approximating 0.04 ml per N . americanus. This falls within the range of results for whole blood loss determined by erythrocyte tagging. Areekul et al. (1971) measured clearance rates of 51Cr (chromic chloride-labelled albumin) in 15 subjects and reported a direct relationship between egg counts and faecal SLCrexcretion rates, and an inverse correlation between serum albumin concentration and faecal isotope losses. Whether pathological loss of plasma exceeds the rate of loss of whole blood (as calculated from clearances of 51Cr-labelled erythrocytes), as proposed by Gilles et al. (1964) and Areekul et a/. (1971), is not clear and will not be established until simultaneous double-labelling techniques are used. In the comparative field there is evidence that pathological plasma losses may exceed figures for loss anticipated from clearance of an erythrocyte label. This has been shown by simultaneous double-labelling experiments in Fasciola hepatica-infected rabbits where the site of loss is not intestine but bile duct (Dargie et a/., 1968; Dargie and Mulligan, 1971). Plasma losses exceeding anticipated losses, from clearances of an erythrocyte label, were also observed from calves infected with Oesophagostomum radiatium (Bremner, 1969), and from geese infected with Amidostomum anseris (Enigk et al., 1969). Under conditions of adequate nutrition (adequate nutrition is exceptional in hookworm-endemic areas) increased intestinal plasma circulation and consequent hypercatabolic albumin metabolism in hookworm-infected people may not necessarily induce significant hypoproteinaemia and hypo-

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albuminaemia. Plasma albumin from pathological intestinal exudation and enterorrhagia are digested in the same way as plasma albumin of the normal exogenous catabolic process. Some of the resultant amino acids will be absorbed and recycled by the liver. Hypoalbuminaemia in hookworm infection follows when the rate of loss exceeds the rate of restoration. The anaemia of chronic hookworm infection, like any anaemia, interferes with the ability of liver cells to synthesize albumin (Ball, 1966). The proportion of the albumin loss that is reabsorbed and reutilized is not, however, known. Absorption depends on the site of the leak and of haemorrhage in the alimentary tract since the lower the position of the worms the smaller will be the amount reutilized. Plasma proteins could not be removed fast enough by experimental chronic bleeding of dogs fed on a protein-rich diet, to exceed the potential of the liver for albumin resynthesis (Robscheit-Robbins et al., 1945), but this does not appear to be the usual situation in abnormal gastrointestinal loss. Jarnum (1963) proposed that under conditions of excessive gastrointestinal protein loss, in various pathological conditions of the alimentary tract particularly idiopathic hypoproteinaemia (at that time plasma loss in hookworm infection was apparently not recognized), a conservative mechanism operated since rate of albumin synthesis was not increased to match the loss. When rate of degradation (or loss) trebled, rate of albumin synthesis increased by only 50 % and serum albumin levels were halved. Similarly, Waldmann (1966) and Ball (1966) stated that, in patients with pathological gastrointestinal protein loss, the maximum rate of albumin synthesis was equivalent to only double the normal rate. Subjects affected by idiopathic hypoproteinaemia lose only protein, whereas hookworm infection results in loss of whole blood and perhaps additional plasma protein from leak lesions. The strain on protein metabolism in hookworm disease is therefore potentially more severe since protein is required for synthesis of both albumin and haemoglobin. Reduced or negative nitrogen balances in severe chronic hookworm infections are common since excess faecal nitrogen derived from intestinal haemorrhage and albumin exudation may offset or exceed dietary intake and absorption. Reduced nitrogen balance is further aggravated since most severe hookworm infections occur in backward or developing countries where dietary protein is in short supply and is often of poor biological value. This leads naturally to discussion of diet and nutrition in hookworm disease. E.

DIGESTIVE FUNCTION AND ABSORPTION

Aspects of nutrition, digestive function and absorption of nutrients in hookworm disease have received considerable attention. There is a concensus that hookworm-infected patients do not appear to be suffering from gross malnutrition, other than iron deficiency (Ashford and Gutierrez-Igaravidez, 1911; Fulleborn et al., 1928; Foy and Kondi, 1960; Sheehy et al., 1962; Layrisse and Roche, 1964; Roche and Layrisse, 1966). Wherever malnutrition is common in hookworm-endemic areas infected subjects (except perhaps those with very large burdens) d o not appear to be less adequately

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nourished than their uninfected or lightly infected counterparts. Variation and depression of appetite have been described (Ashford, 1900; Smith, 1904; Ashburn, 1932; Myrhe and Wallace, 1956; St Martin and Dussault, 1957; Borrero et al., 1961 ; Ball, 1966), and appear to be related to abdominal pain associated with migration and maturation of newly acquired worms. Depravity of appetite and pica, exhibited as geophagia, are apparently rather frequent in chronic hookworm anaemia. Gastric acidity has received considerable attention, particularly in relation to absorption of dietary iron which in hookworm-infected iron-deficient anaemic subjects has prime significance. A tendency in hookworm-infected anaemic subjects to achlorhydria or hypochlorhydria, in basal secretion and/or after histamine or alcohol stimulation, was noted frequently (Garin et al,, 1930; Rhoads et al., 1934; Chavarria et at., 1945; Arora, 1951; Fayez and Ragheb, 1959; Razzak and Hassaballa, 1963; Bajpai and Gupta, 1966; Goyal et al., 1968; Pimparkar et al., 1970). However, a number of these and other reports described normal acid secretion (Chevallier and Brumpt, 1939 ; Yenikomshian and Shehadi, 1943; Bonnin and Moretti, 1950; Bajpai and Gupta, 1966; Goyal et al., 1968). Hyperchlorhydria has also been noted (Yenikomshian and Shehadi, 1943; Bajpai and Gupta, 1966; Goyal et al., 1968). In iron-deficient subjects with hypochlorhydria or achlorhydria, it is not clear which is primary and which is sequential since hypochlorhydria is common in anaemic iron-deficient subjects without hookworm infection (Wintrobe, 1961; Naiman et al., 1964). Razzak and Hassaballa (1963) proposed that severe anaemia (haemoglobin less than 40 %), not hookworm infection, was the cause of hypochlorhydria and reduction in gastric tonus and motility. Gastric enzymtic function has not been investigated in hookworm infection. Intestinal digestion and absorption have received considerable attention. Procedures used include standard tests for absorption of fat, carbohydrate (d-xylose), vitamins A and B,z, and folic acid. Nitrogen balance has also been estimated. Review of the literature on malabsorption in hookworm infection reveals a relatively clear-cut division of findings into two contradictory camps. Absorption is not commonly impaired in uncomplicated hookworm infections, even in most severe infections (Abdalla et al., 1963; Layrisse et al., 1959, 1964; Layrisse and Roche, 1964; Gilles et al., 1964; Salem and Truelove, 1964; Kotcher et al., 1966; Banwell et al., 1967; Ghitis et at., 1967; Mayoral et al., 1967; Aziz and Siddiqui, 1968; Beker, 1971). Alternatively, malabsorption occurs and may be frequent although it is probably not very severe (Saravia et at., 1962; Sheehy et al., 1962; Chuttani et al., 1967; O’Brien and England, 1966; Tandon, 1968; Tandon et al., 1969; Teotia et al., 1969; Nath et al., 1971;Devakul et al., 1970). In the latter group, it is noteworthy that many malabsorption findings resembled tropical sprue, and many of these reports originated in areas (i.e. India, Puerto Rico) in which tropical sprue is relatively common or endemic. Normal absorption (i.e. compared with uninfected control subjects in the same population) has been reported in areas in which tropical sprue is less frequent or rare (Africa, Egypt, Venezuela). This divergence between sprue and non-sprue endemic

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areas also extends to findings of histological abnormalities in the intestinal epithelium (reviewed below). On balance, the evidence appears to favour the theory that malabsorption does not contribute significantly to morbidity, nor is it necessarily a consequence of hookworm infection. Malabsorption in sprue-endemic areas is probably associated with sprue and not with hookworms. In spite of this consensus, it is difficult to explain observations by Sheehy et af. (I962), O’Brien and England (1966) and Burman et af. (1970) that partial remission of malabsorption signs may follow anthelmintic treatment, unless there is synergism between subclinical sprue and superimposed hookworm infection. Enterorrhagia and plasma exudation in hookworm infection may alter intestinal ecology so that conditions favour sprue-like sequelae, perhaps as a consequence of deleterious changes in the microflora. Further information separating malabsorption from hookworm infection was provided by Ghitis et af. (1967) and Falaiye et a/. (1974), who showed that malabsorption in hookworm-infected subjects was a consequence of intercurrent primary protein malnutrition. Naiman et a/. (1964) demonstrated also that the results of most absorption tests in subjects with severe iron-deficiency anaemia, but without past or present hookworm infection, were abnormal. The evidence is therefore confusing, although it appears to favour the hypothesis that malabsorption is not a direct consequence of hookworm infection in man. Very little has been described on secretion and activity of digestive enzymes in hookworm infection. A single report (Gupta et a)., 1973) showed reduced intestinal lactase activity in subjects with iron-deficiency anaemia; nevertheless, findings were similar irrespective of the aetiology of the anaemia (i.e. whether associated with hookworm infection or other causes). This field has been more thoroughly investigated in experimental systems (Symons and Fairbairn, 1962, 1963; Symons, 1969; Symons and Jones, 1970; Gallagher et af.,1971). Reductions in secretion of enzymes originating from or associated with epithelial cells, particularly with their brush borders, were noted in parasitized areas of the duodenum and ileum in rats infected with Nippostrongylus brasiliensis. Enzymes responsible for esterification of long-chain fatty acids were also reduced. The connection between this system and digestive function in hookworm-infected people is tenuous. Moreover, although specific enzyme activities in infected areas of the rat intestine were depressed, these deficiencies were adequately compensated by unaffected areas (Symons, 1969). There was, consequently, no overall depression of protein digestion (Symons and Jones, 1970). Gross and microscopic pathological changes in the intestinal mucosa of N . brasiliensis-infected rats were also more severe than those found in human hookworm infection. Malaviya and Sindhe (1960) claimed to have demonstrated depressed tryptic activity in duodenal secretions in human hookworm infection, and thereby proposed that the aetiology of hookworm anaemia and hypoproteinaemia was a primary protein malnutrition through malabsorption ; an hypothesis not supported by the literature. There is no evidence that anaemia of hookworm infection is related to primary protein deficiency or malabsorption, nor are the parameters of

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hookworm anaemia characteristic of protein deficiency (Roche and Layrisse, 1966). High protein diets achieved only transient respite in the development of hookworm anaemia in heavily infected subjects (Rhoads et al., 1934). Where malnutrition is established in a hookworm-endemic population high protein diets may nevertheless cause temporary improvement in lightly infected subjects (Medal and Hernandez, 1952). Absorption of nitrogen was unaffected in light infections (Holmes and Darke, 1959), although a statistically significant increase in faecal nitrogen was observed in severely anaemic subjects. These authors therefore assumed that heavy hookworm infections reduced apparent digestibility and absorption of energy and nitrogen (and hence they hypothesized on the significance of “anti-enzymes’’ secreted by hookworms). This is reminiscent of findings in ruminant parasitism where depression in apparent digestibility of proteins with apparent decrease in nitrogen absorption is well established. Recent findings, discussed above, may serve to explain poor or negative nitrogen balance in gastrointestinal helminthiases, since nitrogen in the faeces may be derived from whole blood spilled by haematophagus hookworms, and/or from plasma from albumin leakage that occurs in many alimentary helminthiases, including haematophagus hookworms. Although findings of a negative or poor nitrogen balance are real, they are probably unrelated to digestion and absorption of dietary protein. Location of the worm population is important in determining whether faecal nitrogen is increased, depending on the ability of parasitized and inferior (or caudal) segments of the intestine to absorb amino acids from degraded plasma protein exudates and/or whole blood. Evaluations of intestinal motility, peristalsis and their visualization after a barium meal with fluoroscopic and radiographic observation in hookworm infection have shown coarsening and thickening of mucosal folds with an irregular feathery pattern, excessive peristalsis, flocculation, puddling and increased segmentation in the distal duodenum and jejunum (Krause and Crilly, 1943; Yenikomshian and Shehadi, 1943; Hodes and Keefer, 1945; Urso and Mastrandrea, 1954; Sheehy et al., 1962; Salem and Truelove, 1964; Rowland, 1966; Banwell et al., 1967; Chuttani et a/., 1967). The severities of the changes varied widely and in severe cases were similar to duodenal ulceration. Aetiology of the radiological abnormalities has been variously attributed to toxins from the worms, to localized allergic reactions, and to damage to the intramural nerve plexus and its function. The abnormalities were usually quickly resolved after removal of the worms. In some endemic sprue areas the radiological irregularities, like malabsorption, only partially diminished after removal of the hookworms (Sheehy et a/., 1962; Burman et al., 1970), suggesting that perhaps intercurrent subclinical sprue may accentuate this aspect of hookworm pathophysiology. Another important facet of the relationship between diet, morbidity and mortality in hookworm infection is the potential interaction between diet and immunity. In this context the effect of immunity (or more precisely resistance) should be separated into two components, immunity to infection, and resistance to morbidity and mortality of infection. The connection between diet

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and resistance to morbidity and mortality has been a constant theme with specific emphasis on iron intake and iron balance. There is no statistically valid evidence in man that diet or deficiencies therein have an effect on immunity or resistance to hookworm infection (Tripathy et al., 1971). Nevertheless, such a relationship is not excluded, particularly in the worst conditions of protein-calorie malnutrition of children, under which Smythe et al. (1971) described a “nutritional thymectomy” exhibited by wasting of lymphoid tissue, atrophy of the thymus and reductions in delayed hypersensitivity reactions and lymphocyte transformation. Some findings in the early dog-hookworm literature indicate that gross malnutrition may be important in depressing immunity to infection. F.

SECONDARY EFFECTS ON ORGAN FUNCTION REMOTE FROM THE WORMS

A variety of secondary pathophysiological defects have been described in organs remote from the hookworm. The connections between some of these and the primary and secondary pathogenesis of hookworm infection are not difficult to ascertain, the most obvious being mediated by severe anaemia and cellular hypoxia. Many of these secondary remote effects have been discussed above, and the evidence for the aetiological connections of others is discussed below. Renal function in hookworm infection has received considerable attention, not because of specific hookworm-related renal dysfunction, but rather in an attempt to link hypothetical renal defects with the oedema observed in severe chronic hookworm infections. There are no proven connections between renal function and this oedema, which is a consequence primarily of hypoalbuminaemia. The literature that proposes a connection between renal function and oedema in hookworm infection is entirely review and does not contain original protocols to support the hypothesis (Wickramasuriya, 1937; Arora, 1951). There is, on the other hand, substantial data to show that renal function is not affected by hookworm infection (Brumpt and Sang, 1955; Abdalla et al., 1963). Brumpt and Sang (1955) reported high urinary output and rapid cure of oedema accompanying severe chronic hookworm anaemia after anthelmintic treatment. There are few reports on liver function in hookworm infection and the scope of the investigations has been restricted by superficial and insensitive procedures. Most authors concluded that liver function was normal (Brumpt and Sang, 1955; Abdalla et al., 1963; Gilles et al., 1964; Razzak, 1965; Pimparkar et al., 1970), although Gilles et al. (1964) recorded some abnormal bromsulphthalein clearances in severely anaemic patients. Yamasaki and Saruta (1954) claimed to have detected liver damage (unspecified) and Fowler et al. (1968) reported hepatomegaly in severe hookworm anaemia with peripheral oedema. Fatty infiltration is common in anaemia and necropsy appearances of livers in anaemic subjects, both human and canine, have been described as abnormal. It would not be unexpected if, in cases of severe anaemia, some of the more sensitive liver function tests were found to be abnormal. Gilles et at. (1964) and Ball (1966) suggested that cellular hypoxia in chronic severe anaemia (e.g. haemoglobin less than 6 g 100 ml-*) and

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resultant biochemical inefficiency may contribute to the aetiology of hypoalbuminaemia in hookworm infection by impairing reserve capacity for albumin synthesis. Splenomegaly has been described in hookworm infection (Arora, 1951 ; de Azevedo, 1965) although this relationship is probably fortuitous depending on the co-occurrence of malaria and hookworm. Other abnormalities in physiology have been reported although the connection between many of these observations and hookworm infection or anaemia is often tenuous and may perhaps be coincidental. These include hypoparathyroidism (de Azevedo, 1965), retinal haemorrhages (Anon, 1964; Borrero et al., 1961), slow healing of wounds (Stiles, 1912), enlarged parotid glands (Lehmann, 1949; de Azevedo, 1965; Woodruff, 1965). Many of these may be consequences of protein malnutrition, rather than anaemia. Allen and Dean (1965) described kwashiorkor or marasmus-like signs in children infected with hookworms. Although hookworm is probably of secondary significance, increased exogenous catabolism of plasma albumen via hookworm-induced intestinal exudation will aggravate these conditions.

VIII. PATHOLOGY Most descriptions of human hookworm pathology have been derived from gastrointestinal biopsy specimens and only a small amount from necropsy aaterial; the latter, almost invariably of very severe infections, is in older literature and includes little detailed histopathology. AT THE SITE OF LARVAL PENETRATION

A.

There is no information on pathological changes in the human alimentary tract after oral infection. Information on the pathological consequences of percutaneous infection, other than the voluminous literature on clinical symptoms and signs, is similarly rare. It is necessary for detailed histopathology to examine comparative systems, in which differences in nature and degree of skin reactions between hookworm-naive and hypersensitive subjects may be observed. B.

ON ROUTE OF LARVAL MIGRATION

There is no substantial information on the pathological consequences of larval migration through the lungs of man, although this is well described in various experimental systems. D’Abrera (1958) incriminated hookworm, along with other nematode parasites having a similar migratory phase (e.g. ascarids and filarial worms), as a cause of tropical pulmonary eosinophilia, and described foreign body reactions with giant cells, macrophages and fragments of larvae ( N . americanus). From results in the A . caninum-dog system (Miller, 1971), it is probable that in man pulmonary damage and reactions to migrating larvae will be of two types; first, mechanical disruption caused by escape of larvae from capillaries to alveoli with local or extensive areas of alveolar haemorrhage that (in dogs) may prove fatal and secondly, an

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allergic/immune reaction, possibly similar to D’Abrera’s description, in which migrating larvae of a challenge infection are attacked by the sensitized immunological system. In the dog-hookworm system, the lungs appear to be the major site of antigenic stimulation and of effective immunological reaction to subsequent invading larvae (Miller, 1965, 1966b, 1971). It is unlikely in man that events of the first category would be recognized since the acute/peracute course, fatal termination and necropsy would require infections of at least one million larvae for a child and three to four million for a susceptible adult. These figures are extrapolated on a body weight basis from known lethal primary infections in dogs and other experimental animals. There exists one necropsy report (Zimmerman, 1946), reminiscent of the acute and peracute canine diseases that describes multiple recent small haemorrhages with larval debris in the lungs of a 2-year-old child that died from acute ancylostomiasis. There is no information in man on changes in other tissues through which hookworm larvae may pass during their migration (e.g. regional lymph nodes, upper respiratory tract and oesophagus), except for one report (Floch and Thomassen, 1963), which describes diffuse/mild to atrophic gastritis. It is, however, not clear that the lesions in the gastric mucosa were attributable to hookworms (e.g. an immunological reaction to migrating larvae), to anaemia and/or iron deficiency, or to concurrent sprue or malnutrition, since removal of the worms and correction of the iron deficiency did not improve the histological picture. C.

INTESTINAL PATHOLOGY

There are many descriptions of gross and histopathological changes in the jejunum (where most adult hookworms settle), acquired by peroral capsule biopsy techniques. The literature consensus is divided, as it was in relation to malabsorption, between those who believe that hookworm infection induces severe diffuse atrophic jejunal changes (Sheehy et al., 1962; Salem and Truelove, 1964; Tandon et d., 1966, 1969; Rai et al., 1968; Burman et al., 1970; Vieira, 1970; Nath et al., 1971) and those who describe inconspicuous circumscribed mild lesions that regress rapidly after removal of the hookworms (Gilles et al., 1964; Chaudhuri and Saha, 1964; Layrisse et al., 1964; Roche and Layrisse, 1966; Rowland, 1966; Guha et al., 1968; Brandborg, 1971 ; Beker, 1971). Banwell et al. (1964), Chuttani et al. (1967) and Aziz and Siddiqui (1968) reported that atrophic jejunitis was common in the general population, including hookworm-infected subjects, and therefore discounted any relationship to hookworm infection. Severe atrophic changes in the jejunal mucosa, moreover, did not resolve quickly or completely after removal of the hookworms by anthelmintic treatment. “Normal” mucosal structure of indigenous controls was often abnormal (by accepted standards for advanced countries), hookworm-infected subjects did not usually show more severe changes even in areas where tropical sprue is uncommon (Banwell et al., 1964), and the severity of the pathology was not usually related to intensity or to other parameters of hookworm infection. Mayoral

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et al. (1967) and Falaiye et al. (1974) showed that malabsorption and histological abnormalities in hookworm-infected subjects were a consequence of long-standing protein malnutrition and not attributable to the infection. The consensus therefore is that conditions such as malnutrition and sprue are primary aetiological agents for the more severe structural and functional changes in small bowel mucosa; these changes may be exaggerated by superimposed hookworm infection. The general picture in uncomplicated human hookworm infection includes small punctiform haemorrhages or erosions in the mucosa (1-2 mm diameter) with engorgement of local capillaries and some oedema. Infiltrations of lymphocytes, plasma cells and eosinophils in the submucosal tissues are a constant finding. The more severe and diffuse changes, suspected to be of mixed aetiology, ranged from mild villous atrophy with reduction in the villous/crypt ratio, to partial or subtotal atrophy with obliteration of a major proportion of normal villous structure. At the most severe end of the spectrum, blunting and fusing of villi and complete loss of normal stru-t ure was observed. Such extreme severity was, however, rare. To confuse the issue further, Naiman et al. (1964) showed that iron-deficiency anaemia (without hookworm) may cause mild malabsorption with similar mucosal histopathology, but Rawson and Rosenthal (1960) reported no changes in the same circumstances. Functional acquired immunity against hookworm infection in man is at present conjectural (see below). Histological changes typical of immune reactions, including “self-cure” immune worm expulsive phenomena (e.g. cellular infiltration with lymphocytes, plasma and mast cells, oedema and some alteration in the epithelial structure), have nevertheless been recorded in biopsy and necropsy specimens from infected subjects. D.

EXTREME PATHOLOGY IN FATAL ACUTE HOOKWORM INFECTION

The pathological changes in fatal cases, described in older literature (Ashford and King, 1907; Whipple, 1909; Schapiro and Nauck, 1931; Zimmerman, 1946), are much more severe than those observed in biopsy material. Atrophic changes in the jejunal mucosa were not mentioned in the older post-mortem reports. Detailed necropsy reports of fatal hookworm infection in the past 20 years are conspicuously absent. Most recent necropsy descriptions are of chronic hookworm infection ; pathological changes attributable to larvae were therefore not observed. Severe intestinal pathology was recorded by Zimmerman (1946) observing overwhelming acute hookworm infection of infants. He described acute jejunitis and jejuno-ileitis with ulceration, severe haemorrhage, suppuration, necrosis and gangrene and, in more than 25 %, fibrino-purulent peritonitis. Many of his subjects were also afflicted with terminal pneumonias and/or pulmonary oedema with circulatory collapse. Histopathological findings included marked thickening of the intestinal wall by oedema with cellular infiltration. There were multiple petechial haemorrhages in the jejunal mucosa which was infiltrated, as were the oedematous submucosal tissues,

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with neutrophils and eosinophils. More severe changes included dense infiltration of the entire jejunal wall, extensive severe and massive intraluminal haemorrhage. Abscesses were seen in the submucosal tissues, into which the heads of individual hookworms were observed to have penetrated. Other authors have also described penetration of the mucosa by worms, with their subsequent inclusion in either an abscess (Elmes and McAdam, 1954) or in submucosal blood-filled cavities or blood cysts. The pathology and possible significance of the latter have already been discussed. Zimmerman (1946) frequently observed thrombosis and inflammation of the submucosal blood vessels leading occasionally t o gangrenous changes. Other authors (Ashford and King, 1907; Whipple, 1909; Schapiro and Nauck, 1931) described necropsy findings of a more chronic nature in adults. Their gross and histopathological findings were similar to the less severe biopsy changes reported in some recent literature (Gilles et al., 1964; Chaudhuri and Saha, 1964; Layrisse et al., 1964; Roche and Layrisse, 1966; Rowland, 1966 ; Brandborg, 1971), and included multiple petechial haemorrhages, some submucosal oedema and a variable degree of cellular infiltration. E.

PATHOLOGY IN ORGANS REMOTE FROM THE HOOKWORMS

Necropsy changes in tissues remote from the adult hookworms appear to fall into three different aetiological categories. These are the physiological response to haemorrhage and anaemia in bone marrow, the immunological response to the worms in the retroperitoneal nodes and the physiological response to anaemia and cellular hypoxia in the circulatory system, liver and kidneys. The most important of the remote histopathological changes attributable to hookworm infection is the reaction of bone marrow to haemorrhage, anaemia and iron deficiency. Bone-marrow histology is based more on biopsy material in severe chronic anaemia than on necropsy examinations of fatal cases. Since there have been no significant new observations or hypotheses subsequent to the literature review by Roche and Layrisse (1966) only a brief synopsis is necessary. The prime finding is, of course, absence of ironstained haemosiderin. Wide variations occur in levels of erythropoietic activity. The general consensus is of a moderate erythroblastic hyperplasia with erythroblast/myeloblast ratios close t o unity. Fatty changes and inadequate regeneration have been recorded in adults. Extramedullary erythropoiesis has been described in children with megakaryocytes in pulmonary capillaries, spleen and liver (Zimmerman, 1946). Some of the contradictory findings (e.g. hypoplastic marrow, megaloblastic proliferation) have been shown to be consequences of concurrent deficiencies (e.g. folic acid, vitamin BIZ)or biochemical defects secondarily induced by iron deficiency (Roche and Layrisse, 1966). The only change in the granulocytic series was an increase in eosinophils, as is common in many helminth infections. The mesenteric or retroperitoneal lymph nodes have invariably been noted to be enlarged, succulent and hyper-reactive (Ashford and King, 1907; Schapiro and Nauck, 1931; Zimmerman, 1946; Tokumo, 1956). Tokumo

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(1956) described the lymph nodes as oedematous and hyperaemic with dilation of lymphatics, increased reticulum cell proliferation and some infiltration by eosinophils and plasma cells, most of the increased activity being in enlarged germinal centres. This reaction is related to antigenic secretions of attached hookworms in the intestine, and perhaps also to secondary bacterial invasion by opportunistic bacterial pathogens. That these changes were associated with proliferation of immunologically competent lymphocytes and other related cells was shown in the dog-hookworm system through successful transfer of protective immunity and hypersensitive skin reactions by cells harvested from these tissues (Miller, 1967a). Histopathological changes in the third category include chronic passive congestion of the viscera with hypertrophy and dilation of the heart, ascites, hydropericardium, hydrothorax, anasarca and pulmonary oedema (Yates, 1901; Ashford and King, 1907; Schapiro and Nauck, 1931; Zimmerman, 1946). Sommers (1959) described pulmonary oedema and atelectasis in fatal acute congestive cardiac failure in chronic hookworm infection. Anaemia and cellular hypoxia and/or hypoalbuminaemia may be responsible for fatty degeneration of liver, kidneys and myocardium with centrilobular necrosis in the liver, and some nephrosis. Early authors attributed these changes to hookworm infection (Yates, 1901; Ashford and King, 1907; Schapiro and Nauck, 1931) but this cannot be accepted without reservation in view of the widespread use at that time of toxic anthelmintics (e.g. thymol and beta-naphthol). IX. IMMUNOLOGY The necessity of differentiating between immunity or resistance to reinfection and resistance to, or tolerance of the effects of an infection has been emphasized above. The latter aspect has already been discussed. This section therefore reviews immunological reactions in man acquired as the result of previous exposure to hookworms or to their antigenic fractions. These reactions have been separated into two categories, diagnostic and protective. The first concerns serological or host tissue reactions that may have potential diagnostic value but for which evidence of a functional protective action is lacking. In the second subsection the evidence for the occurrence in the manhookworm system of a functional protective resistance to reinfection is discussed. A.

DIAGNOSTIC IMMUNOLOGY

The relative ease of diagnosis by faecal examination has had a suppressive effect on development of other diagnostic methods. Additionally, in apparent absence of substantial evidence that functional protective immunity occurs in the man-hookworm relationship, impetus to investigate reactions and mechanisms of immunity in vitro and in vivo has been lacking. Most literature has described experiments that have been superficial, repetitive and lacking in detailed protocols, and the results have not led to any major practical

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developments nor have they thrown much useful light on the host-parasite relationship. The greater part of this literature describes intradermal tests with various hookworm antigen preparations, and demonstrates abundantly that infected subjects exhibit immediate hypersensitive reactions. Sensitivity may persist for several years after expulsion of worms by anthelmintic treatment (Noda, 1953; Shih-Huei et af., 1959; Yamanaka, 1960). Antigens included protein (Sawada et al., 1954a) or polysaccharide fractions (Noda, 1953; Sawada et al., 1954a) prepared, respectively, by tryptic digestion of larvae or adult hookworms (Wei and Kuo, 1958; Shih-Huei et al., 1959; Prasad and Mathur, 1962) or by aqueous extraction (Yamanaka, 1960; Ishizaki et a/., 1961; Prasad and Mathur, 1962; de Hurtado and Layrisse, 1968; Lobel et a/., 1968). Antigens have been prepared from third-stage larvae (de Hurtado and Layrisse, 1968; Lobel et a/., 1968; Ball et al., 1971) and/or from adult worms (Vattuone, 1933; Vendramini and Magaudda-Borzi, 1955; Shih-Huei et a/., 1959; Yamanaka, 1960; Sawada et al., 1961; Prasad and Mathur, 1962). The species of hookworm used to prepare antigens did not appear to matter since positive reactions in man were obtained with antigens prepared from A . duodenafe (Vattuone, 1933; Noda, 1953; Vendramini and MagauddaBorzi, 1955; Wei and Kuo, 1958; Yamanaka, 1960; Prasad and Mathur, 1962), N . americanus (Bachman and Rodriguez-Molina, 1932 ; Yamanaka, 1960; Ishizaki et a/., 1961; de Hurtado and Layrisse, 1968; Lobel el al., 1968) or the canine hookworm A . caninum (Sawada el al., 1954b, 1961; Yamanaka, 1960). There is no qualitative or quantitative information on different antigens since they have not been tested simultaneously. It seems likely that many antigens within the hookworm group may be common to human and animal species (Yamanaka, 1960; Williams, 1970). Antigens of third-stage infective larvae of different- species may be at least as closely related and reactive as are antigens between larval and adult stages within each species. The results show that 80% or more of currently infected subjects have significant reactions (i.e. the sensitivity was 80% or greater). Up to 20% of tests in non-endemic areas gave false-positive readings (i.e. specificity was low). In endemic areas therefore the intradermal tests may be useful in screening for hookworm infection (past or present). Some results indicated that intensity of reactions in skin tests may be related to intensity of hookworm infection. De Hurtado and Layrisse (1968) and Lobel et al. (1968) showed a positive relationship between intensity of skin reaction (diameter of wheal) and level of infection (faecal egg count). This relationship was, nevertheless, imprecise and modified by age and sex of the subjects. Lobel et al. (1968) found that their intradermal tests were more sensitive than coprological methods for detecting light infections. It is recognized, however, that when the objectives of treatment programmes are less than complete eradication of hookworm infection, it is only necessary to treat the heavily infected subjects and those exhibiting anaemia. Neither of these parameters can be satisfactorily identified by skin tests. Ishizaki et al. (1961) showed a significant correlation between wheal size, eosinophilia and the presence of hookworm signs and

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symptoms, but no relationship to the occurrence and severity of anaemia. Yamanaka (1960) observed statistically significant correlations between the results of intradermal and precipitin tests. False-positive test results in non-endemic and in hookworm-free areas raise the question of specificity. Assuming that the antigens used were sterile, they have, nevertheless, been prepared from adult worms (probably in a state of decomposition) collected from stools after anthelmintic treatment, or from infective larvae cultured on faecal material. Both original materials would be contaminated heavily with enteric and faecal microorganisms. Residual antigens from these micro-organisms may be responsible for false-positive reactors (up to 20 %) where hookworm infection is unknown (Bachman and Rodriguez-Molina, 1932). Persistent false positives after successful treatment may be similarly explained in hookworm-endemic areas (Bachman and Rodriguez-Molina, 1932). Exposure of the human population to infective larvae of canine and feline hookworms would be likely also to reduce specificity. Other immunological reactions have been elicited by inoculation of hookworm larvae and/or their antigens. Harada (1962) induced Arthus, Prausnitz Kustner, Schultz Dale, Schwartzman and guinea pig anaphylaxis reactions in an investigation of a syndrome (Wakana disease) that includes asthmatic cough and vomiting consequent to consumption of fresh or boiled green vegetables in a hookworm-endemic area. A wide range of serological reactions in vitro have also been shown to be mediated by hookworms and their antigens, including precipitin (Yamanaka, 1960; Ball and Bartlett, 1969), haemagglutination (Desowitz, 1967; Jayewardene and Wijayaratnam, 1968; Ball and Bartlett, 1969), complement fixation (Usami, 1919; Magaudda-Borzi and Pennisi, 1958; Ball and Bartlett, 1969), gel diffusion (Singh, 1965), latex flocculation (Rombert et al., 1967) and specific immunofluorescence (de Azevedo et al., 1971). Ball and Bartlett (1969) described a longitudinal study of immunological reactions (in the senior author) to experimental infections with N . arnericanus. He exhibited many of the classical immunological reactions (e.g. positive fluorescent antibody test to larval antigens, haemagglutination titres and P-K reactions) but still appeared to be susceptible, based on worm egg counts, to reinfection and superinfection. B.

FUNCTIONAL PROTECTIVE IMMUNITY

It is clear that live hookworm larvae and somatic antigens derived from larvae and adult worms are capable of inducing, in experimental animals and in man, a variety of serological reactions, usually associated in other systems (e.g. bacterial, viral and protozoal) with protective immunity. There is no conclusive evidence, however, that man develops a functional immunity to hookworms. Acquired immunity with a protective function, nevertheless, has been found to be universal in other hookworm-host systems, when efforts have been made to discover and investigate the subject. In species other than man, success in proving immunity has depended eventually on the ability to infect experimentally, kill and recover worms, In this respect the medical,

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compared with the veterinary, research worker is at a great disadvantage. Controlled procedures such as worm recovery after treatment are nevertheless available where volunteer experimentation is permitted. Since these facilities do not appear to have been widely utilized, one can only search for indicators of protective immunity in epidemiological literature and in the results of a few experimental infections reported in older publications. Unfortunately, little of this material includes valid control data. In a number of epidemiological surveys age-related incidences and intensities of hookworm infection show clearly that worm burdens increase rapidly in the first 10 years of life, remain constant or are reduced in the next 20-30 years and sometimes rise again in old age. Few of the authors incriminated immunity as a factor (Fulleborn, 1929; Cort, 1932; Charmot and Reynaud, 1962; Dammin, 1962; de Azevedo et al., 1971) although this pattern clearly supports an hypothesis that age resistance and/or acquisition of functional immunity are at least contributory factors. Co-existence of a high prevalence of skin reactions to invading larvae, soil severely contaminated with infective larvae, and low morbidity and mortality in the adult population (Cort, 1932) are clear indicators to immunological control of a dangerous situation. Further support for the hypothesis occurs in the results of mass treatment and subsequent acquisition of new hookworm burdens. After anthelmintic treatment the rate of reinfection decreased as number of worms increased so that after 2-3 years the intensity of infection amounted to only 50-60% of pretreatment levels (Docherty, 1926; Hill, 1926). Reinfection rates in children under 10 years old were exceptional since they quickly re-acquired large burdens (Hill, 1926). In the absence of limiting factors (such as immunity) and under suitable climatic and behavioural conditions for reinfection, the rate of increase in burdens -should rationally be an exponential function progressing to severe and overwhelming disease, instead of the observed inverse function. Further indirect evidence of immunity is provided by statistical analyses (Li and Hsu, 1951) of frequency distributions of incidence and intensity of a number of helminth infections in populations of people and animals. The shapes of the curves were similar for both A . duodenale infection in man, and A . caninum infection in dogs, indicating that the same controlling factors were probably operating in both systems. Under conditions favourable for larval development and infection the principal factor controlling epizootiology and disease in the A . caninum-dog system is immunity. It is reasonable to assume that acquired immunity also plays a major part in the similar epidemiology of A . duodenale in people. It may be argued that high incidences of hookworm infection in man, even though worm burdens are small, indicate that acquired immunity is not an important factor. This situation is not, however, incompatible with immunity since in most host-parasite systems with strong host immunity, epizootiology has a similar pattern. Absolute sterile immunity with complete destruction of every challenge larva is unusual. High incidence and low intensity with no acute disease in members of a population (e.g. in A . caninurn-

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infected dogs or A . duodenalelN. americanus-infected people) are certainly not an indicator of susceptibility. If faecal egg counts in adult dogs were to be similarly adopted as proof of absence of immunity to A . caninum infection very misleading conclusions would be reached (Miller, 1978). Without a conscientious treatment programme, almost every dog in hookwormenzootic areas shows evidence of infection; adults have lower (up to 5000 eggs g-l), and younger susceptible dogs much higher faecal egg counts. Only very young pups are severely affected and deaths from canine ancylostomiasis are rare in pups over 3 months. Without experimental evidence that immunity confers almost complete protection against the pathogenesis of challenge worms (Miller, 1971, 1978), it might have been assumed similarly on circumstantial evidence that dogs do not develop functional protective immunity. Small challenge hookworm burdens in the “immune” man may, over an extended period (years), prove more serious than in the immune dog, which can acquire sufficient dietary iron from the most unaesthetic sources to sustain the minimal blood loss from their incapacitated challenge infection. The diet of chronically, but even lightly infected human subjects, is usually so iron deficient that they are unable to compensate indefinitely for even small blood losses. It has been inferred (Duvoir el al., 1942; Beaver, 1945; Brumpt, 1952; Ball, 1966; Ball and Bartlett, 1969) that ability to re-establish infections in volunteers with A . duodenale and N . americanus is evidence of lack of functional protective immunity in man. The apparent negative results of these uncontrolled experiments can be explained by observations made during the development of the canine hookworm vaccine (Miller, 1978). Their findings neither support nor negate an hypothesis of immunity in the man-hookworm relationship. Most of their primary infections, or widely spaced reinfections, comprised only 50-300 larvae, too few and too infrequent to stimulate effective immunity. The minimum immunogenic dose in a 2-4 week doublevaccination schedule, with vastly more immunogenic irradiated larvae in the dog hookworm vaccine, exceeds these numbers. Their uncontrolled challenge infections were similarly extremely small, probabIy below the minimum threshold required to trigger an effective immune response. In man, histological reactions to invasion of hookworm larvae and to adult worms are similar to those seen in the canine hookworm system, and are typical of immunological reactions to helminth parasitism in other systems, consistent with, and indicative of, functional immunological reactions. Hyperactivity of retroperitoneal lymphoid tissue in human hookworm infection is highly reminiscent of the canine hookworm system in which it has been shown possible to transfer, from vaccinated donors to hookwormnaive recipients, functional immunity (including delayed hypersensitive reactions) by lymphoid cells harvested from these hyperactive nodes (Miller, 1967a). There is no histological evidence to suggest that man is incapable of reacting with functional immunological responses to infection with hookworms. Substantial evidence of functional protective immunity in the hookwormman relationship is the most important remaining void in our knowledge of

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the diseases caused in man by hookworms. This author is convinced, from the circumstantial evidence above and from results of self-experimentation (Miller, 1975), that immunity in the man-hookworm system is likely to be very similar to that observed in the dog-hookworm and other host-parasite systems. AND VISCERAL LARVAMICRANS X. CUTANEOUS Cutaneous and visceral larva migrans encompass a variety of conditions resulting from invasion, migration and perhaps encystment of larvae of foreign parasites within the human (and animal) body. In hookworms the cutaneous aspect (larva migrans or creeping eruption) predominates in recognized incidence and severity over the visceral aspect. The first description of cutaneous larva migrans was 100 years ago (Lee, 1875). For the next 50 years the aetiology remained obscure. Kirby-Smith et a / . (1926, 1929), Dove (1927), Shelmire (1928) and White and Dove (1928) rectified this situation by showing that these persistent lesions were caused by invasion of the skin by third-stage nematode larvae, primarily of hookworm. Aetiological emphasis has been on A . braziliense, although other species of animal hookworm have also been shown responsible. The early lesions resemble the reactions after infection by the proper human hookworms, A . duodenale and N . amevicanus. Cutaneous larva migrans then develops the classical picture with advancing tortuous inflammatory tracts within the dermis, accompanied by intense itching. Fortunately this condition is selflimiting and disappears within a few weeks. Three species of hookworm can cause creeping eruption, A . braziliense (Kirby-Smith et a / . , 1926, 1929; Shelmire, 1928; White and Dove, 1928), U. stenocephala (Fulleborn, 1926, 1927), and Bunostonum phfebotomum (Mayhew, 1947). This order relates to more frequency of reports incriminating each species and not necessarily to relative world-wide importances nor severities of the conditions caused by each species. A . caninum has not been included since the consequences of cutaneous infection with its infective larvae resemble the milder reactions caused by the proper hookworms of man. Brief periods of larval migration with very short tracks have occasionally been attributed to A . caninum and N . americanus, but extensive migration by their larvae is uncommon. The aetiological position of A . ceylanicum is uncertain since after experimental infection of volunteers less than 1 % of larvae induced brief creeping eruption (Maplestone, 1933; Haydon and Bearup, 1963; Wijers and Smit, 1966; Bearup, 1967). This observation and the relatively recent differentiation of A . braziliense and A . ceylanicum as distinct species explain earlier apparent conflicts in the literature between the American school that described creeping eruption but no subsequent intestinal infection ( A . braziliense) and the Asiatic/Australian school that recorded little or no cutaneous larval migration and subsequent patent infection with “takes” of up to 75 % ( A . ceylanicum). De Carneri and Castellino (1964) reported that A . tubaeforme did not cause skin lesions in man. but it was not clear if larvae of A. tubaeforme can

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penetrate human skin. Repeated infections may also be necessary to establish hypersensitivity before skin lesions develop. It would not be surprising if the aetiology of many non-migrating lesions is not recognized, since lesions following penetration of human skin by A . caninum larvae are very similar to those caused in sensitized people by mosquitoes and culicoides. The significance of prior infection and development of hypersensitivity in the occurrence, severity and persistence of skin lesions has not been investigated. It is the experience of the author that skin lesions in man with A . caninum did not develop at first exposure. The severity of skin reactions appeared to be greater (i.e. persistence and intensity of itch, oedema, papule development and serum exudate) the longer one had worked with this hookworm and hence the greater one’s immunological experience with larvae. Dissension exists (Beaver, 1956, 1959, 1966) over proof that natural field cases of cutaneous larva migrans can be attributed to A . braziliense since it is extremely difficult to obtain skin biopsies with any certainty that a single larva will be in a place, position and condition for serial sections to reveal all its anatomical parts (White and Dove, 1928). There is also little information to permit adequate differentiation between the larvae of the common hookworm species in histological material. Most evidence for aetiology of naturally occurring cutaneous larva migrans is therefore circumstantial. Nevertheless, the original observations of White and Dove (1928, 1929), Shelmire (1928), and Kirby-Smith et al. (1926) on lesions caused by A . braziliense and A . caninum, and on their differentiation, satisfied many of Koch’s postulates. The same applies to experimental or accidental induction of cutaneous larva migrans by U . stenocephala (Fulleborn, 1926, 1927) and B. phlebotomum (Mayhew, 1947). For further details on geographical distribution, incidence, clinical signs and gross and microscopic pathology etc., the review by Donaldson et al. (1950) is probably the most comprehensive available, although its geographical terms of reference are rather restricted. Visceral larva migrans caused by hookworm larvae has not been so widely recognized, although larvae of A . caninum and/or A . braziliense can reach human pulmonary tissue and cause pneumonitis (Wright and Gold, 1946; Muhleisen, 1953). Temporary slight pulmonary damage, demonstrable radiologically, accompanied by vague chest pains, mild cough, malaise and low-grade fever were observed after extraordinarily massive accidental infection of man with A . caninum larvae (unpublished observations by author). In mice and other abnormal hosts, canine and feline hookworm larvae accumulate and remain alive in the tissues (Nichols, 1956; Soh, 1958; Miller, 1970b). It is therefore reasonable to assume that after penetrating human skin, many hookworm larvae may remain viable in man, who probably served in a prehistoric food chain also as a potential transport host for hookworms of carnivores, as does the mouse today (Miller, 1970b). Hookworm larvae, described as being “Ancylostoma type”, have been described in association with small corneal opacities (Baldone et al., 1964; Nadbath and Lawlor, 1965). It is not clear whether larvae in this situation result from direct superficial infection of the eye from the environment, or from larvae migrating from some deeper tissue.

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Card, R. T. and Weintraub, L. R. (1971). Metabolic abnormalities of erythrocytes in severe iron deficiency. Blood 37, 725-732. Carr, H. P. (1926). Observations upon hookworm disease in Mexico. American Journal ofHygiene 6 (July Suppl.), 42-61. C.C.T.AJW.H.0. (1963). “African Conference on Ancylostomiasis.” World Health Organisation, Technical Reports Series 225, Geneva. Chandler, A. C. (1929). “Hookworm Disease-Its Distribution, Biology, Epidemiology, Pathology, Diagnosis, Treatment and Control.” MacMillan, New York. Chandler, A. C. (1955). “Medical Parasitology.” Wiley, New York. Charmot, G. and Reynaud, R. (1962). Les desordres erythrocytaires au cours de I’ankylostomiase. Medicin Tropical 22, 7-1 7. Chaudhuri, R. N. and Saha, T. (1964). Jejunal mucosa in hookworm disease. American Journal of Tropical Medicine and Hygiene 13,410411. Chavarria, A. P., Blanco, R. P., Herrera, C. S. and Carvajal, E. C. (1945). Influencia de la acidez gastrica en el metabolismo del hierro de las anemias secundarias graves de la malaria y anquilostomiasis en el nino. Revista Medica de Costa Rica 6, 383-389. Chevallier, P. and Brumpt, L. (1939). L‘estomac dans l’anemie ankylostomique et quelques remarques sur cette anemie. Sang 13,331-334. Chowdhury, A. B. and Schad, G. A. (1972). Ancylostoma ceylanicum: a parasite of man in Calcutta and environs. American Journal of Tropical Medicine and Hygiene 21, 300-301. Chuttani, H. K., Peiri, S. K. and Misra, R. C. (1967). Small intestine in hookworm disease. Gasteroenterology 53, 381-388. C.G.O. (1959). “The Status of World Health.” Committee on Government Operations, U.S. Govt. Printing Office, Washington. Conrad, M. E., Weintraub, L. R., Sears, D. A. and Crosby, W. H. (1966). Absorption of hemoglobin iron. American Journal of Physiology 211, 1123-1 130. Cort, W. W. (1932). Variations in hookworm disease. Journal of Parasitology 19, 142-147. Crowley, L. V., Pollack, H. and Brockett, J. E. (1956). Studies on nutrition in the Far East. XIII. The relation of intensity of hookworm infection to changes in body weight and clinical signs of nutritional deficiency. Metabolism 5, 297-301. Cruz, W. 0. (1934). Pathogenesis of anaemia in hookworm disease. 11. Causes which determine regenerative and degenerative phenomena in this anaemia and contributions towards the elucidation of their innermost mechanism. Memorias do Instituto Oswaldo Cruz 29, 427-485. Cruz, W. 0. and de Mello, R. P. (1948). Prophylaxis of hookworm anemia deficiency disease. Blood 3, 457464. D’Abrera, V. St. E. (1958). The aetiology of “tropical eosinophilia”, with a preliminary note on the pathology of the syndrome. Ceylon Medical Journal 4, 195210. Daftary, V. G. and Bhende, T. M. (1956). Anaemia in ancylostomiasis. Journal of Postgraduate Medicine 2, 44-50. Dammin, G. J. (1962). Hookworm Disease. In “Principles of Internal Medicine” (T. R. Harrison, ed.), pp. 1214-1216. McGraw-Hill, New York. Dargie, J. D. and Mulligan, W. (1971). The onset and development of anaemia and hypoalbuminaemia in rabbits infected with Fasciola hepatica. Journal of Comparative Pathology 81, 187-202. Dargie, J. D., Holmes, P. H., MacLean, J. M. and Mulligan, W. (1968). Further studies on the anaemia in fascioliasis. Simultaneous use of W r labeled red cells and 95Nblabeled albumin. Veterinary Record 81, 360-361.

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