Cryptosporidium and cryptosporidiosis

Article about cryptosporidium and cryptosporidiosis.

Protozoan parasite Cryptosporidium which causes the parasitic infection Cryptosporidiosis

Topics covered:

  • Essentials
  • Introduction
  • Biology
  • Molecular biology
  • Epidemiology
  • Zoonotic transmission
  • Human-to-human transmission
  • Waterborne transmission
  • Foodborne transmission
  • Nosocomial transmission
  • Demography
  • Clinical aspects
  • Laboratory investigations
  • Differential diagnosis
  • Treatment of cryptosporidiosis
  • Laboratory detection and diagnosis Infectivity, resistance, and control
  • Further reading

Essentials

Cryptosporidia are small coccidian parasites that infect the mucosal epithelia of a variety of vertebrate hosts, including humans, affecting the health, survival, and economic development of millions of people and animals worldwide. Human infection is mainly caused by two species: (1) Cryptosporidium parvum—also prevalent in young livestock; can be transmitted from animals to humans (zoonotic transmission, particularly important in children), from person to person (‘urban’ cycle, due to faecal–oral spread), through contamination of public drinking-water supplies (which can produce massive outbreaks) or food (prepared by a sick food handler), and nosocomially. (2) C. hominis—essentially a human parasite; may produce large waterborne outbreaks.

Clinical features—infection involves either children or adults, but is a major cause of diarrhoea in children under 5 years old in both developed and developing countries. Patients may be asymptomatic or experience acute or chronic diarrhoea, depending on their age and immune status: (1) immunocompetent humans—infection usually results in acute self-limiting diarrhoea; (2) patients immunocompromised by drugs or AIDS, and those with concurrent infections such as measles or chickenpox—clinical symptoms are more severe and persistent and may become chronic, leading to electrolyte imbalance, wasting and even death.

Diagnosis and treatment—diagnosis is usually made by detection of oocysts in stool, often by use of direct fluorescent-antibody tests. Detection of soluble Cryptosporidium antigens in faecal samples by enzyme-linked immunosorbent assay (ELISA) is useful for the screening of large numbers of specimens. Patients who are immunocompetent are usually managed symptomatically: there is no very effective anticryptosporidial treatment, but those with persistent disease can be given nitazoxanide. Management of patients who are immunocompromised is difficult: aside from supportive care, highly active antiretroviral therapy (HAART) can be effective, both by immune reconstitution (in patients with HIV/AIDS) and by direct inhibition of parasite proteases.

Prevention—primary control is by limiting the opportunity for faecal–oral transmission, both direct and indirect, with maintainence of drinking-water quality and general hygiene (especially in hospitals, wards, etc.) essential for the prevention of the infection. Secondary control, when water supplies are contaminated, can be achieved by boiling water or filtering it (using an appropriate device) before drinking.

Introduction 

The cryptosporidia are obligate intracellular parasites of many vertebrate species. In humans, infection is caused mainly by two species, Cryptosporidium parvum, which is also prevalent in young livestock and can be transmitted zoonotically, and C. hominis, which is essentially a human parasite. First described in laboratory mice by Tyzzer in 1912, Cryptosporidium was recognized as a cause of human infection in 1976. In the 1980s it emerged worldwide as a common cause of severe or life-threatening infection in severely immunocompromised patients, especially those with AIDS, and of acute, self-limiting gastroenteritis in otherwise healthy subjects, especially children.

Biology 

Cryptosporidium species have been traditionally considered as members of the coccidia (phylum Apicomplexa), but recent investigations have revealed a closer phylogenetic affinity with the Gregarinae, which are parasites of invertebrates. The oocyst, containing four sporozoites, is an environmentally robust transmissible stage and is fully sporulated and infective upon excretion with the host faeces. Cryptosporidia are monoxenous, i.e. they complete their lifecycle in a single host. C. parvum is not tissue specific but shows a predilection for the lower ileum during the primary stages of infection. Following ingestion of oocysts, the motile sporozoites are released, through a suture in the oocyst wall, in the lumen of the small bowel. They quickly attach superficially to cells, rounding up to form fixed trophozoites (meronts). The initial site of infection is the brush border of enterocytes in the small bowel, but the parasite is able to infect other epithelial and parenchymal cells. The complex life cycle includes both asexual and sexual stages of replication. The endogenous (tissue) stages develop within a parasitophorous vacuole, the outer layer of which is derived from the host cell’s outer membranes, in a unique intracellular but extracytoplasmic location.

Life Cycle of Cryptosporidium

Above: Diagrammatic representation of the lifecycle of C. parvum. Following ingestion of oocysts, the motile sporozoites are released, attach to cells, and develop into fixed trophozoites (uninucleate meronts) in an intracellular but extracytoplasmic location. These undergo schizogony (asexual multiple budding), the first-stage meronts producing 8 merozoites, some of which recycle to form further type I meronts. Type II meronts produce 4 merozoites, which form gamonts (sexual stages) that mature as either macrogametes or as microgamonts containing 16 motile microgametes. Most of the zygotes formed after fertilization develop into thick-walled, environmentally resistant, transmissible oocysts, which then sporulate, usually by the time they are excreted. Some have only a thin unit membrane, which ruptures to release the sporozoites in situ to produce an autoinfective cycle.

Molecular biology 

The sequences of the genome of both C. parvum and C. hominis have been described and have revealed many peculiar characteristics. The genomes are very compact, about 10 Mb organized in six chromosomes, and are essentially composed of genes. Unusual biochemical pathways and genes have been described and these may serve as novel targets for drug development. Protocols based on nucleic acid amplification of specific genes are available to differentiate Cryptosporidium species and genotypes in both clinical and environmental samples.

Epidemiology 

C. parvum occurs worldwide and is common in humans and in young livestock animals, especially lambs and calves, and has been reported in goats, horses, pigs, and farmed deer as well as in mammalian wildlife. Prevalence in humans varies both geographically and temporally. Because of the diversity of host species that can infect humans, the epidemiology of the infection is complex and involves both direct and indirect routes of transmission from animals to man (zoonotic transmission) and from person to person (urban cycle). 

Cryptosporidium Taxonomy - Oocysts of C. parvum and some C. parvum-related species.

Above: Oocysts of C. parvum and some C. parvum-related species.

Zoonotic transmission 

Transmission from livestock is common, particularly in children, including those from urban homes and schools visiting educational farms and rural activity centres. Companion animals have long been considered potential sources for human cryptosporidiosis. However, they appear to be most commonly infected with host-specific nonzoonotic Cryptosporidium species; they are therefore not considered important reservoirs of infection. Cryptosporidiosis is rarely seen in adults in rural areas, presumably as a result of frequent exposure and the development of immunity.

Human-to-human transmission 

Cases of human-to-human transmission have been reported between family members, sexual partners, children in daycare centres, and hospital patients and staff. Outbreaks in daycare centres have been reported in the United Kingdom and the United States of America, mainly as a result of direct (person-to-person) faecal–oral transmission, although the infection may be introduced in the first instance through zoonotic contact. Affected adults may acquire infection from young children in the home or occupationally. Infection may be transmitted sexually where this involves faecal exposure. Cryptosporidium is a cause of traveller’s diarrhoea, although apparently not as frequently as Giardia.

Waterborne transmission

In the United Kingdom, the United States of America, and elsewhere, there have been numerous well-documented outbreaks resulting from contamination of public drinking-water supplies. Outbreaks, which can be massive, have been associated with C. hominis, which indicates contamination of the supply by human sewage, or with C. parvum, which suggests an animal source of contamination. Isolates from endemic (sporadic) cases, some of which will be waterborne, fall into both categories. Oocysts have been demonstrated widely in both raw and treated water and legislation has been introduced in the United Kingdom and the United States of America in an attempt to limit the latter.

Foodborne transmission 

Cryptosporidiosis has been attributed to ingestion of contaminated apple juice, chicken salad, milk, and food prepared by a sick food handler. Food-borne transmission is probably underestimated, because the long incubation period (3–7 days or more) makes the relationship between cryptosporidiosis and a possibly contaminated food item difficult to establish.

Nosocomial transmission 

Transmission has been reported between health care staff and patients and between patients, particularly the immunocompromised. Large numbers of oocysts may be present in patients’ stools and in vomit; transmission via fomites occurs, although this route is limited by the susceptibility of oocysts to desiccation. Poor hand-washing practice has been identified as an important factor. In an outbreak with high mortality in a ward of immunocompromised patients in Denmark, transmission was probably by patients’ hands via a ward ice-making machine.

Demography

Age and sex distribution

In the United Kingdom, approximately two-thirds of cryptosporidium-positive samples are from children between 1 and 10 years of age, with a secondary peak in adults under 45 years; the infection is uncommon in infants less than 1 year old and in older people. Distribution appears to be the same in both sexes. A relative increase in adult cases is often seen in waterborne outbreaks. In developing countries, infection is common in infants less than 1 year old and asymptomatic infection is common in older subjects.

Temporal distribution

In the United Kingdom, a marked bimodal seasonal pattern of disease has been described: one peak during spring and the second during late summer/early autumn. The spring peak, which coincides generally with lambing and calving, is almost exclusively due to C. parvum, while both C. parvum and C. hominis occur in the late summer/early autumn peak. In the United States of America, the peak onset of cryptosporidiosis occurs annually from early summer to early autumn and, as it coincides with the summer recreational water season, it might reflect the increased use of communal swimming venues, particularly by susceptible hosts like young children.

Frequency of occurrence

Laboratory rates of detection in immunocompetent subjects average about 2% in developed countries (range <1–5%) and about 8% in developing countries (range 2–30%), and Cryptosporidium is about fourth in the list of pathogens detected in stools submitted to the laboratory. In the United Kingdom, from about 5000 to 6000 confirmed cases are reported annually; it is generally somewhat less frequent than giardiasis. Among young children in the United Kingdom, cryptosporidiosis is more common than salmonellosis and detection rates may exceed 20% during peak periods.

Cryptosporidiosis is one of the most common causes of diarrhoea in patients with AIDS and in some studies prevalence has exceeded 50%. The infection rate in patients with AIDS in the United Kingdom has been falling in recent years, which has been attributed to infection control advice and the use of multiple antiretroviral therapy. Infection rates are not generally increased for most other immunocompromised groups.

Clinical aspects 

Pathology

Histopathology

There is mucosal involvement of the small bowel, other parts of the gastrointestinal tract, and sometimes beyond. Moderate to severe abnormalities of villous architecture occur, with stunting and fusion of villi and lengthening of crypts. There may be evidence of mild inflammation, with some cellular infiltration into the lamina propria.

The endogenous stages of the parasite in the luminal surface are generally inconspicuous and appear as small (2–8 μm) bodies, apparently superficially attached to the brush border, unevenly distributed over the apical cells, and within the crypts of the villi. Peaking and apoptosis of infected cells have been reported. There is usually little intracellular change at the ultrastructural level beyond the attachment zone of the parasite. Rectal biopsy may reveal mild nonspecific proctitis. Extensive and chronic involvement of the bile duct and gallbladder is seen in some patients with AIDS.

Immunological response

The particular immunodeficient conditions in which cryptosporidiosis has been reported to show increased severity or persistence suggest that both humoral and cellular factors have a role in limiting infection. An immune response has been demonstrated in the main immunoglobulin classes, although the initial IgG response may be poor. Serological diagnostic tests are, however, of little clinical value. Seroprevalence studies indicate that the infection is common, even in developed countries, and this may reflect water supply quality or other exposures.

Reports differ on the effect of breastfeeding on incidence in infancy; some studies suggest a protective effect although protection from the environment by breastfeeding may also be important.

Although functioning humoral and cellular immunity seems to be important in limiting or controlling infection, it currently appears that, in animal models, CD4+ and CD8+ T lymphocytes and interferon-γ are especially important in this respect. In humans, CD4 cell counts of fewer than 200 cells/mm3 probably indicate the need to take special care to avoid exposure to Cryptosporidium, and fewer than 100 cells/mm3 indicates a poor prognosis if infection occurs.

Possible pathogenic mechanisms

The watery diarrhoea is characteristic of noninflammatory infection of the small bowel, especially that associated with toxin-producing organisms and enteric viruses. Several mechanisms have been suggested to explain the symptoms: reduction in absorptive capacity, particularly for water and electrolytes; increase in secretory capacity from crypt hypertrophy; osmotic effects from loss of brush-border enzymes (e.g. disaccharidases) resulting in malabsorption of sugars, increased osmolality of chyme, and subsequent microbial fermentation of sugars in the colon (which may account for the characteristic offensive smell); and toxic activity.

Clinical presentation in otherwise healthy (immunocompetent) people

Cryptosporidiosis in the immunocompetent person is a self-limiting, acute gastroenteritis with a variety of presenting symptoms. In cases where the time of exposure has been known, the incubation period was about 5 to 7 days (range probably 2–14 days; wider limits have been suggested but are unlikely). There may be a prodrome of 1 day to a few days, with malaise, abdominal pain, nausea, and loss of appetite. Gastrointestinal symptoms start suddenly, the stools being described as watery, greenish with mucus in some cases, without blood or pus, and very offensive. Patients may open their bowels more than 20 times a day but more usually 3 to 6 times. Other symptoms include colicky, abdominal pain, especially after meals, anorexia, nausea, and vomiting, abdominal distension, and marked weight loss. Influenza-like systemic effects, including malaise, headache, myalgias, and fever, commonly occur. Gastrointestinal symptoms usually last about 7 to 14 days, but weakness, lethargy, mild abdominal pain, and intermittent loose bowels sometimes persist for up to a further month.

There is no evidence of transplacental transmission but infection during late pregnancy may cause metabolic disturbances in the mother, leading to the infant’s failure to thrive. Failure to thrive has also been observed in older infants and children and may be associated with persistent infection and enteropathy, especially in developing countries.

Reported sequelae include pancreatitis (associated with severe abdominal pain), toxic megacolon, and reactive arthritis. In immunocompetent patients, deaths are rarely attributable to cryptosporidiosis.

Recent studied in the United Kingdom have further demonstrated that the impact of cryptosporidiosis on public health extends beyond that of acute diarrhoeal illness. Notably, an increased risk of nonintestinal sequelae (joint pain, eye pains, recurrent headache, and fatigue) is associated with infection with C. hominis but not with C. parvum.

Clinical presentation in immunocompromised patients

Susceptibility to cryptosporidiosis and the severity of the disease is increased in patients who are immunocompromised as a result of AIDS, hypo- or agammaglobulinaemia, severe combined immunodeficiency, leukaemia, malignant disease, and bullous pemphigoid. Disease susceptibility and severity are also increased during immunosuppressive treatment with cyclophosphamide and corticosteroids, as in patients undergoing bone marrow transplantation, and in children immunosuppressed by measles and chickenpox, especially where there is associated malnutrition. Infection in patients with leukaemia may be unusually severe and has sometimes proved fatal, particularly when associated with aplastic crisis, and may then require modification of chemotherapy to control the infection.

Symptoms of cryptosporidiosis are generally similar to those in immunocompromised patients but often develop insidiously. In those with late-stage AIDS with very low CD4 cell counts or in some other profound deficiency states, diarrhoea may be frequent, profuse, and watery, like cholera. Patients may open their bowels frequently, passing up to 20 litres of infected fluid stool per day; persistent nausea and vomiting is usually associated with severe diarrhoea and suggests a poor prognosis. Associated symptoms include colicky, abdominal pain often associated with meals, severe weight loss, weakness, malaise, anorexia, and low-grade fever.

Cryptosporidial infection in immunocompromised patients may involve the pharynx, oesophagus, stomach, duodenum, jejunum, ileum, appendix, colon, rectum, gallbladder, bile duct, pancreatic duct, and the bronchial tree. Cryptosporidial cholecystitis (presenting with severe right upper quadrant abdominal pain), sclerosing cholangitis, pancreatitis, hepatitis, and respiratory-tract symptoms may occur, with or without diarrhoea. The clinical picture may include other features of HIV infection and there is often coinfection with other pathogens such as cytomegalovirus, Pneumocystis jiroveci, and Toxoplasma gondii.

Patients with less severe impairment of immunity may experience resolution or a more chronic course, with less profuse diarrhoea, sometimes with remission and then recurrence, possibly associated with biliary tract involvement. Except in those patients whose immune suppression can be relieved by stopping immunosuppressant drugs, or, in the case of HIV, intensifying antiretroviral therapy, severe symptoms may persist until the patient dies. This is either as a result of dehydration, acid–base or electrolyte disturbances, and cachexia, from some other opportunistic infection or malignant disease, or a combination of these.

Laboratory investigations

In early acute cases the stools are usually watery, greenish with mucus in some cases, without blood or pus. Peripheral leucocytosis and eosinophilia are found rarely. Serum electrolyte abnormalities will develop in patients who become severely dehydrated. In immunocompromised patients with cryptosporidial cholecystitis, serum alkaline phosphatase and γ-glutamyl transpeptidase levels are raised, while aminotransferase and bilirubin levels may remain normal.

In patients with AIDS, commonly associated infections are with cytomegalovirus and Isospora belli. Mixed infection with Campylobacter, Giardia, and Cyclospora species may be found in immunocompetent patients.

In the bowel mucosa, there is histological evidence of enterocyte damage, villous blunting, and inflammatory-cell infiltration of the lamina propria; cell peaking and apoptosis have been reported. Histopathological appearances of the affected biliary tract resembles primary sclerosing cholangitis. Radiographic abnormalities include dilatation of the small bowel, mucosal thickening, prominent mucosal folds, and abnormal motility and, in the biliary system, dilated distal biliary ducts, stenosis with an irregular lumen, and other changes reminiscent of primary sclerosing cholangitis.

Cryptosporidium parvum - Immunofluorescence

Above: Cryptosporidium parvum - Immunofluorescence

Differential diagnosis 

The absence of blood, pus, cells, or Charcot–Leyden crystals may distinguish cryptosporidiosis from some acute bacterial diarrhoeas and that associated with amoebiasis and isosporiasis. In immunocompetent patients, the symptoms of cryptosporidiosis resemble those of giardiasis or cyclosporiasis. Intense abdominal pain and cramps are generally more common in cryptosporidiosis, but bloating and weakness less common. In immunocompromised patients, especially in those with AIDS, isosporiasis is clinically indistinguishable, but can be diagnosed by finding the organisms in the stool, where Charcot–Leyden crystals may also be found. This infection responds to treatment with co-trimoxazole, as does cyclosporiasis.

Treatment of cryptosporidiosis 

There have been a large number of studies aimed at developing a satisfactory therapy for human cryptosporidiosis, but these investigations have failed to identify a drug of choice. Several groups may benefit from an effective therapy, particularly patients with HIV/AIDS, transplant recipients, patients undergoing cancer chemotherapy, and those with severe malnutrition. 

Since cryptosporidium infection in the immunocompetent person is a self-limiting disease, a symptomatic antidiarrhoeal treatment is usually sufficient. However, in case of persistent disease, the patients should be given anti-cryptosporidium therapy.

Today, the therapy of choice is nitazoxanide (2-acetyloloxy-N-(5-nitro-2-thiazolyl) benzamide), a synthetic agent that has a demonstrated activity against a broad range of parasites as well as some bacteria. In vitro studies showed inhibition of growth at concentrations of less than 10 μg/ml, and studies in adults have shown that single doses of up to 4 g are well tolerated without important adverse effects.

Immunocompromised patients with persistent severe diarrhoea, malabsorption, and other complications may require prolonged palliative treatment. They should avoid excess milk, as lactose intolerance may develop.Parenteral feeding and fluid, electrolyte, and nutrient replacement may be needed. Antiperistaltic agents such as loperamide, diphenoxylate, or opiates may increase abdominal pain and bloating. Antiemetics may be needed for symptomatic relief.

Temporary relief of biliary obstruction has been achieved by endoscopic papillotomy and of cholecystitis by cholecystectomy. Diarrhoea and vomiting may, however, prove intractable. 

Highly active antiretroviral therapy (HAART) is the treatment of choice for cryptosporidiosis in immunocompromised patients, and can be used not only prophylactically but also as a treatment and secondary prophylaxis for established infections. HAART therapy is effective against cryptosporidiosis and acts both by immune reconstitution and direct inhibition of parasite proteases.

Laboratory detection and diagnosis 

The characteristic endogenous stages may be found in histological sections, using light and electron microscopy, but diagnosis is usually by detection of oocysts in stools. Oocysts have also been found in vomit and sputum in some cases, especially those associated with AIDS. The oocysts of C. parvum are spherical or slightly ovoid, about 4 to 6 μm, and appear refractile in wet faecal preparations with a highly refractile inner body, the cytoplasmic residuum; the four sporozoites within may be distinguished with difficulty using special optical systems.

Several conventional stains have been adapted for diagnostic purposes, such as the modified Ziehl–Neelsen method and phenol–auramine fluorescent stain. Direct fluorescent-antibody tests, which detect intact organisms through the use of monoclonal antibodies that label the oocyst wall, are widely used due to their excellent sensitivity and specificity. Detection of Cryptosporidium soluble antigens in faecal samples by an enzyme-linked immunosorbent assay (ELISA) is very easy to perform and particularly useful for the screening of large numbers of specimens, albeit its specificity is limited by cross-reactions with other antigens of parasitic and nonparasitic origin that can generate false positives.

Standardization of approach to screening and of reporting is essential for epidemiological purposes. Ideally, all stool samples from cases of diarrhoea should be screened; restriction, where unavoidable, should be based on age group (see demography) and not on factors such as stool consistency. Concentration of stool specimens is not usually required for diagnosis in acute cases. Fungal spores, yeasts, cysts of Balantidium, sporocysts of Isospora, and oocysts of Cyclospora may readily be mistaken for cryptosporidial oocysts.

Infectivity, resistance, and control 

Infectivity

In studies using monkeys and lambs, the infective dose for C. parvum was fewer than 10 oocysts. In human volunteer studies in the United States of America, the minimum infective dose for C. parvum and C. hominis appeared to be similar (ID50 was 132 and 83, respectively). In contrast to C. parvum, however, C. hominis elicted a serum IgG response in most infected persons.

Resistance and disinfection

Oocysts can survive for several months in a cool, moist environment but are highly susceptible to desiccation, prolonged freezing, and moderate heat (pasteurization temperatures). They are remarkably resistant to most disinfectants and antiseptics, including chlorine at concentrations far greater than those used in water treatment and even to glutaraldehyde under normal use conditions. Some disinfectants may be more effective if used at elevated temperature (37°C or higher). Oocysts are sensitive to 10 volume (3%) hydrogen peroxide, to appropriate levels of ozone, and to medium or high-pressure ultraviolet.

In hospitals, adequate disinfection of faecal contamination or of endoscopes is difficult. If such instruments have been used for patients with cryptosporidiosis, prolonged immersion in glutaraldehyde at a temperature higher than 37°C, or in hydrogen peroxide, after careful cleaning, may be required to ensure safety.

Control of transmission

Primary control is by limiting the opportunity for faecal–oral transmission, both direct and indirect. Symptom-free subjects not in contact with immunocompromised patients can normally be permitted to work if their hygiene is scrupulous. Spread via fomites is possible but this route is limited by the susceptibility of oocysts to desiccation. Patients with AIDS are more susceptible to infection with uncommon species or genotypes and advice may be needed to limit exposure.

Contamination of water supplies is inevitable, even in developed countries, and may be the source of some sporadic cases as well as outbreaks. When a public advisory notice is issued to boil water, raising the water just to boiling point is sufficient. In general, bottled water and water from point-of-use filters are unlikely to contain parasites but may carry an increased bacterial load, the health significance of which is uncertain for the immunocompromised. Patients with AIDS and others who are profoundly compromised should be advised never to drink water that has not been boiled or filtered through a suitable device. Users of filters should remember that these devices may concentrate potential pathogens and care is needed in replacing and disposing of filter elements.

Hospitals involved in the care of profoundly immunocompromised patients should be particularly vigilant in the management of patients with cryptosporidiosis. Long-term arrangements should be made for the provision of safe water for the immunocompromised to avoid difficulties when a notice to boil water is issued.

Further reading

Top

Cacciò SM, Pozio E (2006). Advances in the epidemiology, diagnosis and treatment of cryptosporidiosis. Expert Rev Anti Infect Ther, 4, 429–43.

Casemore DP (1991). ACP Broadsheet 128. Laboratory methods for diagnosing cryptosporidiosis. J Clin Pathol, 44, 445–51.

Coop RL, Wright SE, Casemore DP (1998). Cryptosporidiosis. In: Palmer SR, Soulsby Lord, Simpson DIH (eds) Zoonoses—biology, clinical practice, and public health control, pp. 563–78. Oxford University Press, Oxford.

Hunter PR, Nichols G (2002). Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin Microbiol Rev, 15, 145–54.

Meinhardt PL, Casemore DP, Miller KB (1996). Epidemiologic aspects of human cryptosporidiosis and the role of waterborne transmission. Epidemiol Rev, 18, 118–36.