Paul Prociv

Immune Responses in Hookworm Infections

SUMMARY Hookworms infect perhaps one-fifth of the entire human population, yet little is known about their interaction with our immune system. The two major species are Necator americanus, which is adapted to tropical conditions, and Ancylostoma duodenale, which predominates in more temperate zones. While having many common features, they also differ in several key aspects of their biology. Host immune responses are triggered by larval invasion of the skin, larval migration through the circulation and lungs, and worm establishment in the intestine, where adult worms feed on blood and mucosa while injecting various molecules that facilitate feeding and modulate host protective responses. Despite repeated exposure, protective immunity does not seem to develop in humans, so that infections occur in all age groups (depending on exposure patterns) and tend to be prolonged. Responses to both larval and adult worms have a characteristic T-helper type 2 profile, with activated mast cells in the gut mucosa, elevated levels of circulating immunoglobulin E, and eosinoophilia in the peripheral blood and local tissues, features also characteristic of type I hypersensitivity reactions. The longevity of adult hookworms is determined probably more by parasite genetics than by host immunity. However, many of the proteins released by the parasites seem to have immunomodulatory activity, presumably for self-protection. Advances in molecular biotechnology enable the identification and characterization of increasing numbers of these parasite molecules and should enhance our detailed understanding of the protective and pathogenetic mechanisms in hookworm infections.

Human enteric infection with Ancylostoma caninum: hookworms reappraised in the light of a “new” zoonosis

Recent studies in northeastern Australia indicate that enteric infection with Ancylostoma caninum is a leading cause of human eosinophilic enteritis. Much more frequent accompaniments of this infection are obscure abdominal pain with or without blood eosinophilia, while a large part of the population is probably infected asymptomatically. These conclusions are based on extensive serological investigations in patients and control subjects, as well as 15 cases in which single, adult hookworms were identified in situ in patients. In no case has more than one worm been identified, and none has been fully mature, so the infections have never been patent. Aphthous ulcers of the terminal ileum, caecum and colon have been seen in association with this infection and have also been observed in almost 5% of patients who are colonoscoped in north Queensland. Serodiagnosis has relied on an IgG and IgE ELISA using excretory-secretory antigens from adult A. caninum, but Western blot using these antigens to identify IgG4 antibodies to a protein of molecular weight 68 kDa (Ac68) promises to be more specific and sensitive. However, identical antigens appear to be secreted by the anthropophilic hookworms as well. The clinical, public health and biological significance of these findings are discussed in detail.

Neuro-angiostrongyliasis: unresolved issues.

Angiostrongylus cantonensis, the rat lungworm, probably evolved with its hosts, members of the genus Rattus and closely related species, in south-east Asia. Since its first discovery in rats in China and in a case of human infection in Taiwan, the parasite has been found to infect humans and other mammals across a wide and ever-increasing territory, which now encompasses much of south-east Asia, Melanesia, Polynesia and eastern Australia. It has also established a foothold in Africa, India, the Caribbean and south-eastern USA. This dispersal has been a direct result of human activity, and in some cases has been linked with the spread of the African giant land snail, Achatina fulica. However, this snail is not critical to the extension of the parasites range, as numerous other indigenous molluscan species serve as adequate intermediate hosts; the importance of Achatina to the life cycle may have been over-emphasized. In Australia, the parasite is established along parts of the east coast, and the presence of an indigenous close relative, Angiostrongylus mackerrasae, suggests a long association of the parasite with its local rat hosts, a situation analogous to that of Angiostrongylus malaysiensis in south-east Asia. These three Angiostrongylus species share virtually the same life cycle, but only A. cantonensis has been confirmed to be a human pathogen.

Human Enteric Infection with Canine Hookworms

Since the 1920s, the common dog hookworm, Ancylostoma caninum, has been known to cause cutaneous disease in persons who have been exposed to infective larvae [1]. Sporadic accounts of its occurrence as an adult worm in the human intestine have been reported from the Philippines [2], South America [3-5], and Israel [6]; the occasional adult A. caninum was found among numerous human hookworm specimens (usually Necator americanus) recovered either after anthelminthic treatment or at autopsy. In no case was the parasite implicated in clinical disease or shown to be fully developed and producing eggs. In 1988, a published series of 33 Australian patients with abdominal pain and blood eosinophilia included a patient with ileal obstruction caused by an eosinophilic phlegmon to which a hookworm was attached [7]. Histologic sectioning precluded specific identification of the parasite. Because human hookworm infection, a persistent Third World problem, is not endemic in urban Australia and has virtually disappeared from the aboriginal communities of Queensland [8, 9], it was speculated that the cases had a common cause, possibly a dog hookworm. Shortly afterwards, two other cases of infection with a single adult A. caninum were reported, both detected at colonoscopy and one associated with eosinophilic ileitis [10, 11]. We developed an enzyme-linked immunosorbent assay (ELISA) and Western blot [12, 13] that are helpful in the diagnosis of abdominal pain with blood eosinophilia and indicate that the parasite is a common cause of this syndrome in eastern Australia. We describe six additional cases of human enteric infection with A. caninum and summarize pertinent features, including serologic findings, of all nine Australian cases so far identified. Methods From 1985 to 1992, five patients were diagnosed by us and four others were referred with single hookworm infections. They lived either in Townsville (latitude, 19 degrees S) or Brisbane (latitude, 27 degrees S). In each case, the medical records and biopsy tissue were provided by the attending clinician and pathologist. At least one serum sample collected within 4 weeks of diagnosis and the formalin- or alcohol-fixed worms were also available from all except one patient (patient 1). Measurements Clinical and demographic data were sought from the records and by interview. A complete blood examination, total serum IgE assay, and fecal microscopy were done in commercial or teaching hospital laboratories. Blood eosinophilia was ascribed to a count higher than 0.50 109/L, and the upper normal value for IgE was 288 g/L. Tissue biopsy specimens were examined and reported by histopathologists not associated with the study. Hookworms were examined microscopically by experienced parasitologists and were drawn and measured using standard techniques. Species identification was based on accepted criteria, which included overall length; esophageal length; body width; number of teeth in the buccal cavity; and, in male worms, length of spicules and structural details of the copulatory bursa [14, 15]. Sera were stored at 15C and tested, among numerous control sera, for IgG and IgE antibodies by one of the investigators who was unaware of the patient histories. The ELISAs incorporated excretory-secretory antigens from adult A. caninum [12]. In the Western blots, these antigens were separated electrophoretically on polyacrylamide gels and transferred to nitrocellulose paper, which was then incubated with patient sera diluted 1:10 [13]. Controls were sera from blood donors in the state of Tasmania, where A. caninum does not exist. The cut-off absorbance levels for positivity in IgG and IgE ELISAs were 0.147 and 0.155 optical density units, respectively. Both the IgG and IgE Western blots were reported as positive when a protein band (Ac68) with a molecular weight of 68 kd was recognized. Results All patients were white; six were men and three were women, with a mean age of 48 years (range, 30 to 61 years). Patients 1 to 3 were diagnosed from 1985 to 1990; the others were diagnosed from 1991 to 1993. Each patient lived in a freestanding house with lawns and garden and kept one or more dogs as pets. By gardening or walking outside barefooted, all patients had been potentially exposed to soil contaminated with canine feces. Except for patient 7, none had recently travelled to an area endemic for human hookworm. None had had atopic or immunologic diseases, and none reported cutaneous or respiratory symptoms that may occur with human or zoonotic ancylostomiasis [1]. After removal of the worm, all patients recovered and have remained well (Table 1). Table 1. A Summary of Demographic, Laboratory, and Parasitologic Findings in Nine Patients Infected by Solitary Hookworms* Patients 1 to 3 The clinical features of these patients have been published previously and are summarized in Table 1 [7, 10, 11]. A 61-year-old woman (patient 1) with a 4-week history of recurrent bowel obstructions had a laparotomy with resection of an inflamed ileal segment. A single hookworm was attached to the mucosa and was sectioned for histologic study, which precluded a species identification. Her case was reported to one of us several months after her recovery; serum had not been stored. A 50-year-old tradesman (patient 2) recurrent abdominal pain and a positive fecal occult blood test was diagnosed at colonoscopy. A feeding hookworm, species A. caninum, was removed from an inflamed and ulcerated terminal ileum. A 53-year-old male teacher (patient 3), having colonoscopy for vague abdominal pain and minor rectal bleeding thought to be caused by hemorrhoids, had a single feeding worm, species A. caninum, removed from his rectum. The colon and rectum appeared healthy. Patient 4 A 41-year-old woman from Townsville described three attacks during the preceding 5 weeks of mid-abdominal pain, which aroused her from sleep, lasted a few hours, and did not require urgent care. Her stools were loose and more frequent after each episode but she felt well between attacks. Results of a physical examination, abdominal ultrasonography, and upper gastrointestinal endoscopic examinations were normal. Colonoscopy, completed 20 cm into the ileum, showed two ulcers, 2 mm in diameter, in the cecum. A feeding nematode, 5 cm proximal to the ileocecal valve, was removed with biopsy forceps (Figure 1). Damage to the buccal cavity prevented specific identification of this 8-mm long, male hookworm, with spicules 750 and 785mlong. Tissue obtained from near the worm and the cecal ulcers was excessively infiltrated with eosinophils and plasma cells. Figure 1. Feeding hookworm in ileum (patient 4). Patient 5 A 45-year-old Brisbane man, with mild chronic dyspepsia that had not been investigated, was hospitalized after 24 hours of increasingly severe colic. He was afebrile and his abdomen was distended and tender; radiologic findings showed dilated loops of small bowel with fluid levels. A complete blood examination showed initial neutrophilia (10.25 109/L), with a normal eosinophil count of 0.37 109/L. After early improvement, the signs worsened; a laparotomy was done 36 hours later and showed increased serous peritoneal fluid and a 24-mm, inflamed ileal stricture, which was resected. An 8-mm long, female hookworm, without eggs, was attached to the lumen of the opened segment, but damage to the buccal capsule precluded its specific identification. Histologic findings showed intense eosinophilic ileitis. Mild blood eosinophilia (0.68 109/L and 0.76 109/L) was recorded 4 and 14 days after admission and resolved (0.15 109/L) by 4 weeks. Patient 6 A 30-year-old Townsville woman complained of anorexia, abdominal pains, diarrhea, and a 6-kg weight loss during a period of 4 weeks. She described the pain as a persistent, dull ache with severe exacerbations lasting 5 minutes, often provoked by eating. Her normal bowel habit, three formed stools daily, had increased to 10 liquid movements, often at night; passing stool gave temporary pain relief. A clinical examination was normal, but the blood eosinophil count was 2.96 109/L. The results of a fecal culture and microscopic examination were normal. The colonoscope was passed approximately 20 cm into the ileum. The only abnormal finding was a hookworm feeding in the cecum; it was removed with biopsy forceps and identified as a 10-mm long, adult male A. caninum, with spicules 785 and 800mlong (Figure 2). Random ileal and cecal biopsy samples showed intense eosinophilic inflammation. Three weeks later, her blood eosinophil count (0.26 109/L) was normal. Figure 2. Scanning electronmicrograph (patient 6). A. caninum Patient 7 A 51-year-old Brisbane man had a laparotomy for suspected acute appendicitis. He had presented 3 days after developing generalized abdominal pain, which improved initially but became severe again just before assessment. Six weeks earlier, he had returned from a 3-week vacation in Vanuatu, in the South Pacific, where he had walked barefooted and had observed many freely roaming dogs and extensive environmental fouling with their feces. He bred and trained greyhound dogs professionally, treating his animals regularly with anthelminthic agents. A clinical examination showed maximum tenderness over the right iliac fossa, with absence of bowel sounds. The operative findings were peritonitis, with slightly turbid fluid (culture sterile; cells not identified) and an intensely inflamed ileal segment 60 cm proximal to the ileocecal junction, surrounding a 2-cm thickened area that at first appeared to be a tumor. The ileum was opened to show a small ulcer that, histologically, was intensely inflamed and infiltrated with eosinophils. An 8-mm long, female A. caninum, without eggs, was attached to the center of the ulcer. Patient 8 A 50-year-old Townsville man with atherosclerosis was hospitalized with a 6-hour history of severe abdominal pain coming in waves lasting several seconds. He had noticed a mild inc

Potential role of head lice, Pediculus humanus capitis, as vectors of Rickettsia prowazekii

Since the pioneering work of Charles Nicolle in 1909 [see Gross (1996) Proc Natl Acad Sci USA 93:10539–10540] most medical officers and scientists have assumed that body lice are the sole vectors of Rickettsia prowazekii, the aetiological agent of louse-borne epidemic typhus (LBET). Here we review the evidence for the axiom that head lice are not involved in epidemics of LBET. Laboratory experiments demonstrate the ability of head lice to transmit R. prowazekii, but evidence for this in the field has not been reported. However, the assumption that head lice do not transmit R. prowazekii has meant that head lice have not been examined for R. prowazekii during epidemics of LBET. The strong association between obvious (high) infestations of body lice and LBET has contributed to this perception, but this association does not preclude head lice as vectors of R. prowazekii. Indeed, where the prevalence and intensity of body louse infections may be high (e.g. during epidemics of LBET), the prevalence and intensity of head louse infestations is generally high as well. This review of the epidemiology of head louse and body louse infestations, and of LBET, indicates that head lice are potential vectors of R. prowazekii in the field. Simple observations in the field would reveal whether or not head lice are natural vectors of this major human pathogen.

Occult enteric infection by Ancylostoma caninum: a previously unrecognized zoonosis.

BACKGROUND/AIMS Human disease caused by the canine hookworm Ancylostoma caninum and a high incidence of eosinophilic enteritis have been reported from northern Queensland, Australia. The aim of this study was to evaluate patients with unexplained abdominal pain and a possible association with cryptic infections by A. caninum. METHODS The clinical and demographic features of patients from this region with eosinophilic enteritis (group A1, n = 42), obscure abdominal pain associated with (group A2, n = 105) and without (group A3, n = 84) blood eosinophilia were reviewed and sera were tested against A. caninum excretory-secretory antigens by enzyme-linked immunosorbent assay (ELISA) and Western blot. Four additional patients, two with confirmed A. caninum, had hookworm infection. RESULTS The level of dog ownership in these four groups was 79%-100%, higher than in the local population (P < 0.001). The ELISA tested positive in 71% of A1, 67% of A2, and 30% of A3, versus 8% in controls (P < 0.002). All cases tested were positive on Western blot versus 10% of controls (P < 0.0001). The ELISA values increased with chronicity and decreased during convalescence. CONCLUSIONS We conclude occult human A. caninum infections are common and are characterized by eosinophilic enteritis and obscure abdominal pain with or without blood eosinophilia. The diagnosis can be confirmed by serology.

Characterization and localization of cathepsin B proteinases expressed by adult Ancylostoma caninum hookworms

The hookworm Ancylostoma caninum induces human eosinophilic enteritis (EE), probably via allergic responses to its secretions. Cysteine and metallo-proteinases may be the components of these secretions that elicit hypersensitivity reactions. In order to characterize genes encoding cysteine proteinases (CP) secreted by A. caninum, an adult hookworm cDNA library was constructed and screened with a cloned fragment of a hookworm CP gene. This fragment was obtained using consensus oligonucleotide, CP-gene-specific primers in the polymerase chain reaction. cDNAs encoding two CPs were obtained from the library and sequenced. The first gene, AcCP-1, encoded a cathepsin B-like zymogen CP of 343 amino acids (aa), predicted to be processed in vivo into a mature CP of 255 aa. Closest nucleotide identities were to Haemonchus contortus cysteine protease (61%) and to human cathepsin B (60%). The second gene, AcCP-2, encoded a mature CP of 254 aa, that showed 86% identity to AcCP-1, and 58% and 47% identity to bovine cathepsin B and human cathepsin B, respectively. Rabbit antisera raised against recombinant AcCP-1 reacted with esophageal, amphidial and excretory glands in formalin-fixed, paraffin embedded sections of both male and female adult hookworms, and with an antigen of approx. 40 kDa in Western blot analysis of excretory/secretory products from adult hookworms. Together, these immuno-hybridization results strongly suggest that the CP encoded by the AcCP-1 gene is secreted by hookworms. These are the first reported CP genes from hookworms. Proteinases encoded by these genes may be responsible for the CP activity that we have shown previously to be secreted by adult A. caninum.

Diagnosis by faecal culture of Dientamoeba fragilis infections in Australian patients with diarrhoea

This paper reports the first survey in Australia to use faecal culture to detect Dientamoeba fragilis in patients with diarrhoea. Of 3 different protozoal culture media evaluated on a case of known infection, modified Boeck & Drbohlavs medium was the most suitable. The organism could be grown from faeces stored for up to 24 h at room temperature, but for only 10 h at 4 degrees C. Culture was then used, in combination with microscopy of smears fixed with polyvinyl alcohol and trichrome-stained, to examine single stool specimens from 260 consecutive patients with diarrhoea in the city of Brisbane. D. fragilis was detected in 4 (1.5%) specimens, only 2 of which were positive by microscopy. Other protozoa were found in 36 (13.8%) specimens: Blastocystis hominis in 28 (10.8%), Giardia duodenalis in 4 (1.5%), Endolimax nana in 3 (1.2%), and Entamoeba coli in one (0.4%). One strain of Dientamoeba was cryopreserved in liquid nitrogen and recultured successfully 10 d later. Culture was more sensitive than microscopy in diagnosing D. fragilis infection and the organism, of dubious pathogenicity, was not common in patients with diarrhoea in this community. Prevalence surveys of intestinal protozoan infections should use faecal culture, and specimens should be less than 12 h old and not refrigerated. Dientamoeba strains isolated by culture can be cryopreserved.

Persistent head lice following multiple treatments: Evidence for insecticide resistance in Pediculus humanus capitis

Viable head lice were found on the scalps of two family members following multiple topical insecticide treatments. The possibility of reinfestation had been reliably excluded. Persistent infestation could be diagnosed only after cutting the hair and combing repeatedly, which allowed visualization of juvenile (nymphal) and adult lice. Insecticide‐resistant headlouse infestations are probably much more common than is generally realised and may persist unnoticed, so that more aggressive approaches will be needed to eradicate these ectoparasites from individuals and communities.

Detection of antibodies to secretions of Ancylostoma caninum in human eosinophilic enteritis.

To evaluate the role of canine hookworms in human eosinophilic enteritis (EE) in north-eastern Australia, we tested human sera in an enzyme-linked immunosorbent assay (ELISA) which incorporated antigens of adult Ancylostoma caninum. Sera from the following groups were examined: 10 patients with EE (unexplained recurrent abdominal pain and related symptoms, with peripheral eosinophilia) from Townsville and Brisbane; 2 persons known to be infected with A. caninum and 20 presumed unexposed healthy controls; 20 patients with other gastrointestinal diseases; 20 with other identified parasitic infections; and 20 with atopic conditions. High levels of specific immunoglobulin (Ig) G and IgE antibodies were found in patients with EE but not other gastrointestinal disease. Excretory-secretory (ES) products were more discriminating than somatic antigens in the ELISA and the IgG/ES-ELISA was the most specific; occasional cross-reactions could be explained on epidemiological or parasitological grounds. The IgM-ELISA was neither specific nor sensitive. We conclude that canine ancylostomiasis is a major cause of human EE in north-eastern Australia, and the pathogenesis is based probably on hypersensitivity to antigens secreted by the parasite.