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Echinococcosis: diagnosis and diagnostic interpretation in population studies
Review
Echinococcosis: diagnosis and diagnostic interpretation in population studies Paul R. Torgerson1,2 and Peter Deplazes1 1 2
Institute of Parasitology, University of Zurich, CH-8057, Zurich, Switzerland Ross University School of Veterinary Medicine, PO Box 334, Basseterre, St Kitts, West Indies
Diagnosis is a basic component of population studies on echinococcosis. Other than careful necropsy in animals, there is no perfect gold standard. In the definitive host, techniques for direct parasite identification include copro-antigen and copro-DNA detection. In intermediate hosts, necropsy is typically used. In humans, diagnostic imaging and serology are both widely employed. The use of multiple parallel testing or an additional confirmatory test (or tests) in a diagnostic strategy can overcome the lack of a perfect gold standard. This will yield valuable information at population and individual levels, providing the study is well designed and any shortcomings of the tests are incorporated into the analysis. Here, we discuss analytical approaches to population studies of echinococcosis. Echinococcosis: a global problem Cystic echinococcosis (CE), which is caused by Echinococcus granulosus, has a global distribution [1]. Adult parasites are found in the intestines of dogs. Eggs, passed in faeces, infect a large number of mammalian intermediate hosts including sheep, pigs and cattle. Larval stages (hydatid cysts) develop in the liver and lungs and occasionally other organs. The life cycle is completed when organs containing these cysts are consumed by dogs. Humans are infected directly or indirectly from eggs excreted with dog faeces. Alveolar echinococcosis (AE), which is caused by Echinococcus multilocularis, is a potentially fatal disease that is confined to the northern hemisphere. Foxes are the usual definitive hosts and small mammals are intermediate hosts. Humans, infected from eggs, develop a highly infiltrative metacestode almost exclusively in the liver, which in late stages, metastasizes to other organs. In many parts of the world, both CE and AE are emerging or reemerging zoonoses [2,3]. E. granulosus has been eliminated in Iceland, New Zealand, Tasmania and southern Cyprus [4] through intensive long-lasting intervention, by periodically treating the dog populations with anthelmintics and/or aggressive culling policies of stray dogs. Control of E. multilocularis remains a greater challenge because of its predominant wild animal life cycle in foxes and small mammals. However, pilot programmes in large rural areas in Germany and Japan and defined urban areas in Zurich
(Switzerland) have demonstrated potential to substantially reduce the parasite prevalence in foxes through the long-term distribution of praziquantel-impregnated baits [5,6]. Diagnosis of infection is central to any control programme because this establishes baseline information against which progress to reduce infection levels can be measured. Diagnosis in the definitive host Necropsy The sedimentation and counting technique (SCT) is considered the gold standard (see Glossary) and the most accurate quantitative method for both species of Echinococcus [7]. The intestine is opened and incubated in physiological saline and the intestinal mucosa is scraped with a spatula. The released worms can be retrieved and counted from the sediment using a binocular microscope. Glossary Conditional dependence: two tests are conditionally dependent when the sensitivity (or specificity) of the second test depends on whether the results of the first test are positive or negative among infected (or non-infected) individuals. Latent class models: these models estimate the test sensitivity, specificity and prevalence (i.e. unknown latent classes) using observed test results. If properly structured, they can be used to estimate the true prevalence of disease or infection in the absence of a gold standard or reference test. They can also be used to develop better reference tests. Negative predictive value: this is the probability of an individual being healthy or non-infected given the observed test result. This also depends on the test characteristics and true prevalence within the population. Perfect gold standard: a test which has both a specificity and sensitivity of 1 or 100%. A perfect gold standard accurately predicts disease or infection status in every individual and in every stage of infection. In practice, a perfect gold standard is rarely available. Positive predictive value: this is the probability of an individual being diseased or infected given the observed test result. This depends not only on the test characteristics but also on the true prevalence within the population. Reference test or gold standard: the reference test is usually an imperfect gold standard. It will be the best available test to define the infection or disease status of an individual. The sensitivity and specificity of the test are usually well defined but often fall short of a perfect gold standard. Sensitivity: the proportion of true positives the test detects. A test with a sensitivity of 0.98 or 98% will be positive in 98 of every 100 truly diseased or infected individuals. Specificity: the proportion of true negatives the test detects. A test with a specificity of 0.98 or 98% will be negative in 98 of every 100 healthy or noninfected individuals. Test prevalence: the proportion of individuals in a population that test positive to a diagnostic test at a single time point. Test prevalence is only equal to true prevalence if the test used is a perfect gold standard. True prevalence: the proportion of individuals in a population which have the disease or infection at a single time point.
Corresponding author: Torgerson, P.R. ([email protected]).
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1471-4922/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2008.12.008 Available online 5 March 2009
Review The intestinal scraping technique is a less laborious method for mass screening and has a sensitivity that is 78% of that of the SCT [8,9]. Deep mucosal scrapings (a total of 15 per intestine) are made using microscope slides, and these are squashed into thin layers and examined microscopically, enabling semiquantitative estimation of the worm burden. Safety precautions must be strictly followed when using either of these diagnostic strategies [10]. Purgation Oral administration of arecoline hydrobromide to dogs results in the purgation of intestinal contents after 30– 60 min. This material can be examined for the presence of Echinococcus parasites. This technique was used during the eradication campaign in New Zealand for mass surveillance [11] and has been utilized in many epidemiological studies worldwide. The technique is nearly 100% species specific and has been used for quantitative studies. However, safety precautions during field work and parasite identification in the laboratory are essential and time consuming [10]. Arecoline can also cause serious adverse reactions in dogs. The sensitivity is poor: studies have generally assumed that the sensitivity of the technique is 65% [12]. However, a recent study indicates that it might be as low as 38% for E. granulosus and 21% for E. multilocularis [13]. Nevertheless, it is a potentially useful technique, particularly when evaluating other diagnostic procedures such as copro-antigen or copro-DNA tests, because it can prove the presence of infection. Copro-antigen enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assays (ELISAs) for antigen detection in dog faeces were initially developed independently by several groups. Initially developed for the diagnosis of E. granulosus [14,15], ELISAs for the detection of E. multilocularis subsequently became available [16,17]. Sensitivity of copro-antigen assays is generally good with moderate-to-high worm burdens (>100 worms) but less in animals with low burdens. Copro-antigens can also detect prepatent infections [14,18–20]. Although sensitivity and specificity have often been defined in selected groups of animals (e.g. experimentally infected animals), the actual performance in the field is less certain because of potential crossreactivity with antigens from Taenia species or other helminths. Furthermore, test parameters will vary with the population on which the test is used. In addition, even with high test specificity, the positive predictive value can be poor if used where the Echinococcus prevalence is low. This occurred in Cyprus in a study of the dog population during the final stages of a control programme to eliminate the parasite [21]. However, coproantigen tests remain useful procedures for population studies, provided the potential pitfalls are fully understood, because they can be performed in both living and dead animals. DNA-based tests Highly specific PCR tests have been developed for use on faeces or on eggs isolated from faeces to confirm the presence of Echinococcus spp. [22–26]. Because PCR is costly and time-consuming, it is not suitable for routine
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diagnostic or large-scale purposes. PCR-based diagnosis of intestinal infections should be implemented within a diagnostic strategy. Copro-PCR is best used for confirmatory purposes of copro-antigen-positive samples (Box 1). Alternatively, it can be used as the preferred method for identification of morphologically indistinguishable Taenia and Echinococcus eggs recovered from samples. A considerable problem with working on faeces is that to obtain a good sensitivity, large volumes are required. PCR procedures might also suffer from inhibition by faecal components, which lower diagnostic sensitivity [27]. One approach to overcome these limitations, which is only suitable for patent infections, is to undertake PCR on isolated taeniid eggs [23]. The use of the sieving-flotation method [23] in this context seems to be more sensitive at detecting taeniid eggs than other methods, such as the McMaster method [28]. A study in Kyrgyzstan has indicated that the sensitivity of egg isolation followed by PCR is 78% for E. granulosus infections and 50% for E. multilocularis infections [13]. This could seem low; however, when the length of the prepatent period and life expectancy of the parasite are accounted for then, arguably, the Box 1. Multiple tests Multiple diagnostic tests are often used on populations. This could include a screening test of high sensitivity and a confirmatory test of high specificity or parallel screening using two (or more) tests on the whole population. If tests are conditionally independent, an individual testing positive to both tests has a high probability of being a true positive (see Table I in Box 2). In humans, ultrasound relies on direct visualization of the parasitic cyst and serology relies on the immunological response to the exposure to the parasite. Consequently, these two tests can be regarded as independent. By contrast, in dogs, copro-antigen, copro-PCR and arecoline all directly detect the presence of the parasite. These tests might, therefore, have a dependence structure. In one theoretical scenario, a prevalence of E. granulosus infection is assumed to be 20% and a copro-PCR test with a sensitivity and specificity of 78% and 93%, respectively, is used. Lack of specificity might be due to coprophagia [13]. In addition, a copro-antigen test of sensitivity and specificity of 88% and 95%, respectively [70], is also used, and this is based on proved positive infections as determined with arecoline treatment or negative dogs in a nonendemic area having received antiparasitic treatment. Conditional independence of the tests would indicate a positive predictive value of 0.98 if both tests are positive. This compares to the individual positive predictive values for copro-antigen and copro-PCR of 0.81 and 0.75, respectively. When there is conditional dependence – for example, with a dependence coefficient of 0.01 between the two sensitivities and 0.02 between the two specificities – the predictive value of two positive tests would be only 0.88. In a second theoretical scenario, there could be a low prevalence of infection of 2%, perhaps after a control programme. The specificity of coproPCR would then be virtually 100% because there would be little intestinal passage of eggs owing to coprophagia. Then, conditional sensitivity of the two tests would make only a small difference to the results. In reality, expensive copro-PCR is only likely to be used as a confirmatory test. Therefore, in the first scenario, if tests are conditionally independent, confirmation by PCR indicates a test prevalence of 0.14, compared to a test prevalence of 0.16 with conditional dependence. In the second scenario, the test prevalence is 0.014 for the independent model and 0.015 for the dependent model. Paradoxically, more samples will be confirmed by PCR as positive if the tests are dependent than if the tests are independent. Thus, a good approach would be to have a screening test of high sensitivity followed by a highly dependent confirmatory test of high specificity.
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Review sensitivity for the detection of patent infections rises to 87% and 72%, respectively. Attempts to improve the sensitivity could include repeated faecal sampling and/or using PCR techniques that do not rely on egg isolation, thus detecting prepatent infections [29]. However, the dynamic of coproDNA excretion is dependent on loss of parasite stages (protoscoleces during the first days after inoculation and immature stages at the end of prepatency), whereas coproantigen concentrations are related in this infection period to parasite metabolic activity [29]. Direct detection of DNA in faeces of E. granulosus-infected dogs revealed 15 dogs positive by copro-PCR during the prepatent period of 58 dogs proven to be infected by E. granulosus, indicating a sensitivity of 26% during this phase of the infection [30]. A further important limitation of copro-PCR is that formalin-fixed faecal material is not suitable, owing to DNA degradation. However, faecal material stored in 70% ethanol or between 20 8C and 80 8C can be used [29]. Serology in definitive hosts Serology for detection of intestinal E. granulosus infections in dogs [31] and E. multilocularis infections in foxes has been investigated. However, problems with sensitivity, specificity and previous infections have prevented specific antibody detection being employed in surveillance studies [32–34]. Diagnosis in intermediate hosts Natural intermediate hosts of E. multilocularis are many small mammal species, and necropsy is the usual method of detecting infection in them. Metacestodes that contain protoscoleces can easily be identified microscopically. However, small or calcified fibrotic lesions of 0.5–5.0 mm in diameter can often be efficiently identified by PCR. In an investigation in 889 water voles (Arvicola terrestris) in a high E. multilocularis-endemic area, 277 liver lesions were detected. Of these, 108 were caused by Taenia taeniaeformis and 26 contained E. multilocularis protoscoleces, as identified by classical morphology. From the remaining small lesions, 55 were positive and 106 lesions were negative by E. multilocularis PCR [35]. For ecological studies, the proportion of protoscolex-containing animals and protoscolex numbers are important for parasite reproduction estimations in intermediate host populations. However, the total number of infected animals represents the infection pressure in a rodent habitat. There are increasing reports of AE in domestic dogs and zoo animals [36]. In these cases, diagnosis in the individual live animal is important. Imaging and serological techniques have been developed and are similar to those used in humans. However, as the dog can be both a definitive and an aberrant intermediate host, any serological test should, ideally, distinguish between these two possibilities. Alternatively, serology combined with copro-antigen or copro-DNA tests will indicate the presence or absence of intestinal infections [37]. Pigs with lesions of AE, confirmed by molecular techniques, have also been described [36]. Even though infections in pigs do not contribute to transmission, they are excellent markers for environmental contamination with eggs. Diagnosis for E. granulosus is rarely indicated in individual farm animals, but population surveys are important 166
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in obtaining baseline information about the parasite epidemiology, particularly in respect to a control programme [38]. Generally, diagnosis in sheep is at postmortem [39]. Sheep slaughtered for meat are often young and will inevitably have a lower prevalence than older animals [38–40]. Furthermore, animals likely to fetch the best price might be sent to the abattoir, and these might be animals that are better conditioned with a smaller likelihood of infection. Thus, abattoir studies, especially if they are not age-stratified, can produce substantial underestimates of the true prevalence. Ultrasound examination can be used in sheep to detect hepatic but not lung cysts. However, sensitivity is disappointing. For example, in Tunisia, mass screening of sheep by ultrasound indicated a prevalence of hepatic echinococcosis of 20% [41]. However, sensitivity is disappointing: studies in Kenya indicate that the sensitivity of ultrasound examination for detection of CE in sheep and goats is 54%, with a specificity of 94% [42]. Immunodiagnosis in sheep presents problems of sensitivity and specificity, limiting its use. Indirect haemaglutination, double diffusion and ELISA have all been attempted with little success [43]. Poor specificity is often attributed to crossreactivity with other taeniids (such as Taenia ovis and Taenia hydatigena). Recent studies using hydatid fluid antigen have reported a sensitivity and specificity of 89% and 90%, respectively, based on macroscopic lesions as the gold standard. This test gave good discrimination against ovine cysticercosis, and the specificity was improved using purified fractions of hydatid cyst fluid (S2B) but with a loss of diagnostic sensitivity [43]. Previous studies using hydatid cyst fluid, antigen B (AgB) purified from hydatid cyst fluid or protoscolex antigens have reported sensitivities ranging from 36% to 90% and specificities ranging from 65% to 96% [44–46]. Diagnosis in humans Ethics require accurate diagnoses in population studies so that the best advice regarding appropriate treatment can be given. Diagnoses are complicated in CE and AE coendemic areas, such as the Tibetan plateau and the Baltic States. The human prevalence can be as high as 5% for both CE and AE in some highly endemic areas of the Tibetan plateau [47], but in general, studies have demonstrated prevalences of <2% and often much lower [48,49], even in areas with high animal prevalences. With such low prevalences, the diagnostic specificity becomes important for accurately identifying individuals who might require treatment. For example, consider a diagnostic test that has a specificity of 98% and a sensitivity of 90% in a large survey of 10 000 people with a true prevalence of 1%. There will be 100 individuals who have hydatid disease, of which 90 will test positive. There will be a further 198 (2%) of nondiseased individuals who will also test (false) positive. In this case, the positive predictive value is 31.3% (90/288). Although the probability of having the disease has increased from 1% (pre-test) to 31.3% (post-test), the test-positive individual is still more likely to be healthy than diseased. (This problem is further illustrated in Box 2.) Highly specific tests are required, multiple tests (such as serology and ultrasound) should be used and accurate interpretation of results is vital. The negative predictive
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Box 2. The expected test results in a hypothetical human screening programme For this calculation, a hypothetical cross-sectional study of 10 000 individuals could be undertaken and echinococcosis diagnosed by ultrasound and serology, assuming the tests are conditionally independent. The specificity of ultrasound has been reported to be 0.96 in the detection of abdominal cysts [71]. To take into account pulmonary echinococcosis, the sensitivity could be assumed to be 0.91, although it could be lower. Sensitivity and specificity of recombinant antigen B (rAG138/2) have been reported at 0.83 and 0.98, respectively [72]. In addition, the sensitivity and specificity of serology is assumed to be the same if patients have hepatic or pulmonary echinococcosis, although in reality this is not likely [54,73]. Based on a true prevalence of 5%, 2% and 1% in the screened population, Table I gives the positive predictive values and expected results of the combination of test results given these assumptions regarding the diagnostic test performances. If ultrasound screening is undertaken and serology is only used as a confirmatory test, then with 5% prevalence, 387 cases would be
diagnosed as CE, of which 8 would be falsely diagnosed. There will also be 486 indicated by ultrasound to be CE but negative by serology. If these cases are further investigated or followed up, it should reveal 76 of these as true positives. This would leave 45 undetected cases in the population. If the whole population is also screened with serology regardless of ultrasound test, there would also be 192 cases that are ultrasound negative but serologically positive. Of these, 38 would be true positives. These could be offered a chest x-ray and follow-up. Seven CE cases would still be negative for both serology and ultrasound. The corresponding numbers for the prevalence of 2% and 1% can easily be found in Table I. Unlike animals, the actual diagnosis in individuals participating in a mass screening is important. However, as illustrated above, a precise diagnosis cannot be given, although the probability of infection can be estimated given the test results and estimated population prevalence. Appropriate recommendations on a case-by-case basis must then be made.
Table I. Positive predictive values in a hypothetical human screening programme of 10 000 individuals Test results Tested positive by both tests
Tested positive by ultrasound only
Tested positive by serology only
Tested negative by both tests
True prevalence Positive predictive values Number testing positive True positives Positive predictive values Number testing positive True positives Positive predictive values Number testing positive True positives Positive predictive values Number testing negative True positives
value in this case is 99.9%; thus, a test-negative individual is unlikely to have hydatid disease, further illustrating that accurate investigation of positive test individuals is important. Ultrasound is widely used and can confirm the diagnosis of abdominal echinococcosis and indicate if lesions are active. Further information on the use of ultrasound can be found in many sources (for example, Refs [49–52]). The main disadvantage of ultrasound is that it cannot normally detect pulmonary echinococcosis. In an ultrasound study based on autopsy records of asymptomatic cases of CE, a ratio of 8.3:1 of liver to lung cysts was recorded [53]. This indicates the sensitivity of ultrasound in mass screening can be as low as 89%, even assuming it has a sensitivity of 100% in detecting liver and abdominal cysts. One screening programme that also used a portable x-ray found a prevalence of pulmonary echinococcosis of 1.1% with an overall prevalence of 5.5% [54]. Therefore, although ultrasound for mass screening is generally considered to have a sensitivity of 88–98% and a specificity of 93–100% for abdominal lesions [49,55], the sensitivity for detecting all forms of echinococcosis is somewhat lower. The sensitivity and specificity to detect AE lesions might be comparable to hepatic CE as primary lesions occur in the liver. However, sensitivity could be reduced because slower developing lesions make early diagnosis more difficult and also compromise specificity. Advanced imaging techniques such as nuclear magnetic resonance or computeraided tomography can be used to confirm diagnosis in humans [51,56] for both forms of disease. Unfortunately, in many remote endemic areas, such facilities are not
5% 0.982 387 379 0.155 486 76 0.196 192 38 0.00084 8935 7
2% 0.954 159 152 0.07 454 30 0.09 174 15 0.00032 9213 3
1% 0.911 83 76 0.034 443 15 0.045 168 8 0.00016 9305 1
available. In this case, serological back-up tests might be necessary to give additional information regarding the nature of the lesion detected by ultrasound. Several serological assays have been developed for the diagnosis of human echinococcosis (reviewed in Refs [57,58]). Native crude antigens, such as hydatid cyst fluid or crude E. multilocularis metacestode material (a complex mixture of glycolipids, lipoproteins and carbohydrates), have generally proved to be sensitive for both CE and AE. However, in a variable proportion of AE or CE patients, no specific antibodies could be demonstrated with a variety of tests. This could be due to complex parasite– host interactions modulating the immune system [59,60]. Specificity is a problem with crossreactions with other helminth species [61]. Specificity was considerably increased on the genus or even species level by purification of antigens such as AgB in hydatid cyst fluid of E. granulosus or Em2 in E. multilocularis metacestode material. Various groups have developed diagnostic tests based on recombinant AgB, which produces better results than native AgB (reviewed by Refs [58,62]), and synthetic peptides mimicking defined epitopes have also been investigated. Sensitivity and specificity of tests based on AgB have largely been evaluated on known hydatid disease patients or known healthy individuals, in which sensitivity has varied between 0.45 and 0.92. Specificity is reported as between 0.71 and 1 with crossreactivity against AE, cysticercosis, and Schistosoma and Toxocara parasites [58]. The diagnostic performance of different antigens for the diagnosis of AE has enabled their use in the diagnosis of individual patients and use in seroepidemiological studies. 167
Review Reported sensitivity ranges between 0.86 and 0.97, with a specificity of 0.61 to 1.00 [63]. Furthermore, recombinant Em18 and Em16 antigens are predominantly recognized by sera from patients with active lesions of AE [57]. In addition to clinical echinococcosis, serology detects exposure to the parasite before disease development or without parasite establishment. In a pig model for AE, specific antibody reactions against E. multilocularis- or E. granulosus-derived antigens were detected one month after experimental inoculation. Interestingly, animals with multiple small lesions of approximately the resolution limit of most imaging techniques showed stronger antibody reactions compared with animals with fewer, larger lesions [64]. In Germany and Switzerland, human serological studies have been undertaken in which several subjects had a serological response against highly specific E. multilocularis antigens with undetectable or only inactive lesions [48,65]. Likewise, in China, serological prevalence of both CE and AE among children was reported to be much higher than actual disease prevalence [66]. Some work has been undertaken to develop tests for circulating antigens to diagnose echinococcosis (see, for example, Ref. [67]). Such tests have not been widely used so far. Population studies in the absence of a reference test In animals, only careful necropsy approaches a perfect gold standard. In humans, advanced imaging techniques such as nuclear magnetic resonance or computer-aided tomography can be used to confirm diagnosis [51,56]. Many investigators understand diagnostic test limitations and make adjustments to test prevalences to estimate true prevalences. However, diagnostic tests are often validated on defined populations of animals, such as dogs with proven infection, either experimentally or through necropsy studies or tests with 100% specificity, such as arecoline purgation. Negative populations might include dogs that have been recently treated with praziquantel. In humans, tests might be validated using cases confirmed by surgery or cases that have classic ultrasound images. This gives valuable information on the test performance in that particular group of animals or humans, but caution must be taken when extrapolating to populations for surveillance. Test characteristics often vary with the population investigated (Box 3). When using tests for population studies, these limitations should be considered. There are now modeling techniques that can overcome the problems of undefined or poorly defined test characteristics (Box 3). The population prevalence can be estimated even when there is little a priori information known about the infection status of any individuals in the population or when there is no available gold standard. Such Bayesian techniques were used in population studies of dogs in Spain using serology and copro-antigen tests to define the true prevalence of infection [68]. In human surveillance, this could, theoretically, be a powerful technique, particularly because ultrasound and serology are likely to be conditionally independent. However, because prevalences are typically low, it would be a challenge to find populations of significantly different prevalences to develop this model. 168
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Box 3. The Hui-Walter paradigm For most diseases, there is a lack of a perfect gold standard, yet recent statistical techniques have been developed that can evaluate diagnostic tests and estimate true prevalence in the absence of such a standard. Most of these techniques use latent class models based upon a Bayesian framework [74,75], but maximum-likelihood-based approaches can also be used. True prevalence and the other latent classes of test sensitivity and specificity can be estimated if at least two conditionally independent tests are used on two populations of a priori known different prevalences. If only one population is available, three tests must be used. Hui and Walter [76] state that any combination of several tests (R) and populations (S) can be used to estimate the true prevalence and test characteristics provided, as long as S R (2R 1 –1); that is, R = 3 tests and S = 1, population will suffice. The numbers of degrees of freedom in the data must be equal to or greater than the number of unknown parameters in the model. Adding an additional population, for example with the two-test model, adds three degrees of freedom but only one extra parameter (the prevalence of the second population). If, however, the tests have conditional dependence, then in the case of the two-population two-test model, the problem becomes nonidentifiable because there are now two additional parameters (i.e. the conditional dependence of the sensitivity and specificity of the tests). Even if further populations are added, the problem remains unidentifiable. The same is true if further tests are added that are not conditionally independent [77]. If conditional independence cannot be assumed then prior information regarding some test characteristics is required. This was the approach used in a study in Kyrgyzstan [13]. Arecoline purgation and copro-PCR were used as the two diagnostic tests and the population of dogs was age stratified (E. granulosus) or stratified by risk factors (E. multilocularis). Because the specificity of arecoline purgation was 1, at least two parameters disappear from the model (specificity of arecoline and the conditional dependence of the specificity of arecoline and copro-PCR). This then enabled the model to become identifiable. However, the HuiWalter paradigm also assumes constant specificity and sensitivity across populations of different prevalences. This cannot always be assumed because sensitivity and specificity can vary [77]. In echinococcosis, there might be variations in crossreacting helminths between different populations and, hence, different rates of crossreactivity. The additional factor that needs to be considered is not only the point estimates but also the confidence intervals or degree of certainty. This is affected by both the sample size and the prevalence. A low prevalence means there are few true positives on which to calculate the sensitivity. For example, a copro-antigen test was described as having a sensitivity of 77% on the basis of being positive in 20 of 26 purge-positive dogs [33], but the confidence intervals are 56–91%. However, specificity can often be well defined when the prevalence is low because most individuals are true negatives.
A further important limitation of using test results in epidemiological studies is risk-factor analysis. Such studies define animals or humans who might be at particular risk of echinococcosis in a population. Standard logistic regression analysis will give risk factors associated with the test results and not necessarily the disease status. Thus, in the case illustrated in which the positive predictive value is 31.3% (despite a specificity of 98%), logistic regression analysis without adjustment for true disease status could give more information about the risk factors for false-positive test reactions rather than disease transmission. Bayesian techniques are now becoming available to analyze risk factors for true disease status, rather than the test results (see, for example, Ref. [13]).
Review Concluding remarks Accurate diagnosis of echinococcosis is challenging. In animals, diagnosis is usually required at a population level. Provided that characteristics of diagnostic procedures are accounted for, meaningful results can be obtained, which can be used in epidemiological studies or for assessing progress of control programmes. In humans, there is a need for population studies and the same caveat applies to estimate population prevalences. However, in such studies, it is also essential to optimize decision making for the individual subject, and here, the lack of a perfect gold standard can make this demanding. For the individual who is diagnosed with imaging techniques but who is subsequently found not to have echinococcosis, this is not necessarily a problem because the subject might have some other space-occupying lesion that requires treatment. Furthermore, the prognosis is improving for AE with modern treatment methods [69] and, hence, early diagnosis is likely to improve the treatment outcome. In a situation in which multiple tests (such as serology and ultrasound) are used, care must be taken when tests give conflicting results because there is a possibility that the subject has echinococcosis. Such subjects should be followed up and appropriate action taken. A screening test of high sensitivity should ensure most cases are detected, but confirmation with a highly specific test is then indicated. Surveillance on human populations with serology as the single diagnostic test must be undertaken with extreme caution, if at all. Even with a serological test of high specificity, the positive predictive value will be low because of the low population prevalences.
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in Middle Eastern Countries With Special Reference to Morocco (Anderson, F.L. et al., eds), pp. 72–84, Brigham Young University Print Services Ziadinov, I. et al. (2008) Canine echinococcosis in Kyrgyzstan: epidemiology and transmission analysis incorportaing diagnostic uncertainty. Int. J. Parasitol. 38, 1179–1190 Deplazes, P. et al. (1992) Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes. Parasitol. Res. 78, 303–308 Allan, J.C. et al. (1992) Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans. Parasitology 104, 347–356 Morishima, Y. et al. (1999) Evaluation of coproantigen diagnosis for natural Echinococcus multilocularis infection in red foxes. Jpn. J. Vet. Res. 46, 185–189 Deplazes, P. et al. (1999) Echinococcus multilocularis coproantigen detection by enzyme-linked immunosorbent assay in fox, dog, and cat populations. J. Parasitol. 85, 115–121 Jenkins, D.J. et al. (2000) Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection. J. Parasitol. 86, 140–145 Kapel, C.M.O. et al. (2006) Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats. Int. J. Parasitol. 36, 79–86 Allan, J.C. and Craig, P.S. (2006) Coproantigens in taeniasis and echinococcosis. Parasitol. Int. 55, S75–S80 Christofi, G. et al. (2002) Screening of dogs for Echinococcus granulosus coproantigen in a low endemic situation in Cyprus. Vet. Parasitol. 104, 299–306 Dinkel, A. et al. (1998) Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. J. Clin. Microbiol. 36, 1871–1876 Mathis, A. et al. (1996) An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs. J. Helminthol. 70, 219– 222 Stefanic, S. et al. (2004) Polymerase chain reaction for detection of patent infections of Echinococcus granulosus (‘‘sheep strain’’) in naturally infected dogs. Parasitol. Res 92, 347–351 Trachsel, D. et al. (2007) Identification of eggs of canine taeniids by multiplex PCR. Parasitology 134, 911–920 Abbasi, I. et al. (2003) Copro-diagnosis of Echinococcus granulosus infection in dogs by amplification of a newly identified repeated DNA sequence. Am. J. Trop. Med. Hyg. 69, 324–330 Mathis, A. and Deplazes, P. (2006) Copro-DNA tests for diagnosis of animal taeniid cestodes. Parasitol. Int. 55, S87–S90 Bruzinskaite, R. et al. (2009) Echinococcosis in pigs, and intestinal infection with Echinococcus spp. in dogs in southwestern Lithuania. Vet. Parasitol 160, 237–241 Al-Sabi, M.N. et al. (2007) Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes. Parasitol. Res. 101, 731–736 Lahmar, S. et al. (2007) Screening for Echinococcus granulosus in dogs: comparison between arecoline purgation, coproELISA and coproPCR with necropsy in pre-patent infections. Vet. Parasitol. 144, 287–292 Gasser, R.B. et al. (1988) Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens. Aust. Vet. J. 65, 369– 373 Craig, P.S. et al. (2003) Echinococcosis: disease, detection and transmission. Parasitology 127, S5–S20 Craig, P.S. et al. (1995) Diagnosis of canine echinococcosis: comparison of coproantigen and serum antibody tests with arecoline purgation in Uruguay. Vet. Parasitol. 56, 293–301 Gasser, R.B. et al. (1993) Serum antibodies in canine echinococcosis. Int. J. Parasitol. 23, 579–586 Stieger, C. et al. (2002) Spatial and temporal aspects of urban transmission of Echinococcus multilocularis. Parasitology 124, 631– 640 Deplazes, P. and Eckert, J. (2001) Veterinary aspects of alveolar echinococcosis – a zoonosis of public health significance. Vet. Parasitol. 98, 65–87 Staebler, S. et al. (2006) Serological diagnosis of canine alveolar echinococcosis. Vet. Parasitol. 141, 243–250 169
Review 38 Torgerson, P.R. and Heath, D.D. (2003) Transmission dynamics and control options for Echinococcus granulosus. Parasitology 127, S143– S158 39 Torgerson, P.R. et al. (1998) Modeling the prevalence of Echinococcus and Taenia species in small ruminants of different ages in northern Jordan. Vet. Parasitol. 79, 35–51 40 Torgerson, P.R. et al. (2003) Modeling the transmission dynamics of Echinococcus granulosus in cattle and sheep in Kazakhstan. Vet. Parasitol. 114, 143–153 41 Lahmar, S. et al. (2007) Ultrasonic screening for cystic echinococcosis in sheep in Tunisia. Vet. Parasitol. 143, 42–49 42 Sage, A.M. et al. (1998) Evaluation of diagnostic ultrasound as a mass screening technique for the detection of hydatid cysts in the liver and lung of sheep and goats. Int. J. Parasitol. 28, 349–353 43 Gatti, A. et al. (2007) Ovine echinococcosis I. Immunological diagnosis by enzyme immunoassay. Vet. Parasitol 143, 112–121 44 Yong, W.K. et al. (1984) Comparison of cestode antigens in an enzymelinked immunosorbent assay for the diagnosis of Echinococcus granulosus, Taenia hydatigena and T ovis infections in sheep. Res. Vet. Sci 36, 24–31 45 Ibrahem, M.M. et al. (1996) Echinococcus granulosus antigen B and seroreactivity in natural ovine hydatidosis. Res. Vet. Sci. 61, 102–106 46 Kittelberger, R. et al. (2002) Evaluation of three enzyme-linked immunoabsorbent assays (ELISA) for the detection of serum antibodies in sheep infected with Echinococcus granulosus. Vet. Parasitol. 110, 57–76 47 Budke, C.M. et al. (2004) Use of disability adjusted life years in the estimation of the disease burden of echinococcosis for a high endemic region of the Tibetan plateau. Am. J. Trop. Med. Hyg. 71, 56–64 48 Gottstein, B. et al. (2001) Is high prevalence of Echinococcus multilocularis in wild and domestic animals associated with disease incidence in humans? Emerg. Infect. Dis. 7, 408–412 49 Macpherson, C.N.L. et al. (2003) Application of ultrasound in diagnosis, treatment, epidemiology, public health and control of Echinococcus granulosus and E. multilocularis. Parasitology 127, S21–S35 50 Kern, P. et al. (2006) WHO classification of alveolar echinococcosis: principles and application. Parasitol. Int. 55, S283–S287 51 Bresson-Hadni, S. et al. (2006) Imaging aspects and non-surgical interventional treatment in human alveolar echinococcosis. Parasitol. Int. 55, s267–s272 52 WHO Informal Working Group (2002) International classification of ultrasound images in cystic echinococcosis for application in clinical and field epidemiological settings. Acta Trop. 85, 253–261 53 Larrieu, E.J. and Frider, B. (2001) Human cystic echinococcosis: contributions to the natural history of the disease. Ann. Trop. Med. Parasitol. 95, 679–687 54 Gavidia, C.M. et al. (2008) Diagnosis of cystic echinococcosis, central Peruvian highlands. Emerg. Infect. Dis. 14, 260–266 55 Macpherson, C.N.L. and Milner, R. (2003) Performance characteristics and quality control of community based ultrasound surveys for cystic and alveolar echinococcosis. Acta Trop. 85, 203–209 56 Turgut, A.T. et al. (2007) Unusual imaging characteristics of complicated hydatid disease. Eur. J. Radiol. 63, 84–93 57 Carmena, D. et al. (2007) The immunodiagnosis of Echinococcus multilocularis infection. Clin. Microbiol. Infect. 13, 460–475 58 Carmena, D. et al. (2006) Antigens for the immunodiagnosis of Echinococcus granulosus infection: an update. Acta Trop. 98, 74–86
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Trends in Parasitology Vol.25 No.4 59 Vuitton, D.A. et al. (2006) Survival strategy of Echinococcus multilocularis in the human host. Parasitol. Int. 55, S51–S55 60 Rigano`, R. et al. (2001) Modulation of human immune response by Echinococcus granulosus antigen B and its possible role in evading host defenses. Infect. Immun. 69, 288–296 61 Eckert, J. and Deplazes, P. (2004) Biological, epidemiological and clinical aspects of echinococcosis: a zoonosis of increasing concern. Clin. Microbiol. Rev. 17, 107–135 62 Mamuti, W. et al. (2005) Recent advances in characterization of Echinococcus antigen B. Parasitol. Int. 55, s57–s62 63 Gottstein, B. et al. (1993) Improved primary immunodiagnosis of alveolar echinococcosis in humans by an enzyme-linked immunosorbent assay using the Em2plus antigen. J. Clin. Microbiol. 31, 373–376 64 Deplazes, P. et al. (2005) Experimental alveolar echinococcosis in pigs, lesion development and serological follow up. Vet. Parasitol. 130, 213– 222 65 Romig, T. et al. (1999) An epidemiologic survey of human alveolar echinococcosis in southwestern Germany. Romerstein Study Group. Am. J. Trop. Med. Hyg 61, 566–573 66 Yang, Y.R. et al. (2008) Serological prevalence of echinococcosis and risk factors for infection among children in rural communities of southern Ningxia, China. Trop. Med. Int. Health 13, 1086–1094 67 Devi, C.H. and Parija, S.C. (2003) A new serum hydatid antigen detection test for diagnosis of cystic echinococcosis. Am. J. Trop. Med. Hyg. 69, 525–528 68 Benito, A. et al. (2006) Dog echinococcosis in northern Spain: comparison of coproantigen and serum antibody assays with coprological exam. Vet. Parasitol. 142, 102–111 69 Torgerson, P.R. et al. (2008) Alveolar echinococcosis: from a deadly disease to a well controlled infection. Relative survival and economic analysis in Switzerland over the last 35 years. J. Hepatol 49, 72–77 70 Lopera, L. et al. (2003) Field evaluation of a coproantigen enzymelinked immunosorbent assay for diagnosis of canine echinococcosis in a rural Andean village in Peru. Vet. Parasitol. 117, 37–42 71 del Caprio, M. et al. (2000) Diagnosis of human hydatidosis. Predictive value of a rural ultrasonographic survey in an apparently healthy population. Medicina (B. Aires) 60, 466–468 72 Rott, M.B. et al. (2000) Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation. Acta Trop. 75, 331– 340 73 Moro, P.L. et al. (1999) Field diagnosis of Echinococcus granulosus infection among intermediate and definitive hosts in an endemic focus of human cystic echinococcosis. Trans. R. Soc. Trop. Med. Hyg. 93, 611– 615 74 Black, M.A. and Craig, B.A. (2002) Estimating disease prevalence in the absence of a gold standard. Stat. Med. 21, 2653–2669 75 Dendukuri, N. and Joseph, L. (2001) Bayesian approaches to modeling the conditional dependence between multiple diagnostic tests. Biometrics 57, 158–167 76 Hui, S.L. and Walter, S.D. (1980) Estimating the error rates of diagnostic tests. Biometrics 36, 167–171 77 Toft, N. et al. (2005) Diagnosis diagnostic tests: evaluating the assumptions underlying the estimation of sensitivity and specificity in the absence of a gold standard. Prev. Vet. Med. 45, 139–162
Echinococcosis: diagnosis and diagnostic interpretation in population studies Paul R. Torgerson1,2 and Peter Deplazes1 1 2
Institute of Parasitology, University of Zurich, CH-8057, Zurich, Switzerland Ross University School of Veterinary Medicine, PO Box 334, Basseterre, St Kitts, West Indies
Diagnosis is a basic component of population studies on echinococcosis. Other than careful necropsy in animals, there is no perfect gold standard. In the definitive host, techniques for direct parasite identification include copro-antigen and copro-DNA detection. In intermediate hosts, necropsy is typically used. In humans, diagnostic imaging and serology are both widely employed. The use of multiple parallel testing or an additional confirmatory test (or tests) in a diagnostic strategy can overcome the lack of a perfect gold standard. This will yield valuable information at population and individual levels, providing the study is well designed and any shortcomings of the tests are incorporated into the analysis. Here, we discuss analytical approaches to population studies of echinococcosis. Echinococcosis: a global problem Cystic echinococcosis (CE), which is caused by Echinococcus granulosus, has a global distribution [1]. Adult parasites are found in the intestines of dogs. Eggs, passed in faeces, infect a large number of mammalian intermediate hosts including sheep, pigs and cattle. Larval stages (hydatid cysts) develop in the liver and lungs and occasionally other organs. The life cycle is completed when organs containing these cysts are consumed by dogs. Humans are infected directly or indirectly from eggs excreted with dog faeces. Alveolar echinococcosis (AE), which is caused by Echinococcus multilocularis, is a potentially fatal disease that is confined to the northern hemisphere. Foxes are the usual definitive hosts and small mammals are intermediate hosts. Humans, infected from eggs, develop a highly infiltrative metacestode almost exclusively in the liver, which in late stages, metastasizes to other organs. In many parts of the world, both CE and AE are emerging or reemerging zoonoses [2,3]. E. granulosus has been eliminated in Iceland, New Zealand, Tasmania and southern Cyprus [4] through intensive long-lasting intervention, by periodically treating the dog populations with anthelmintics and/or aggressive culling policies of stray dogs. Control of E. multilocularis remains a greater challenge because of its predominant wild animal life cycle in foxes and small mammals. However, pilot programmes in large rural areas in Germany and Japan and defined urban areas in Zurich
(Switzerland) have demonstrated potential to substantially reduce the parasite prevalence in foxes through the long-term distribution of praziquantel-impregnated baits [5,6]. Diagnosis of infection is central to any control programme because this establishes baseline information against which progress to reduce infection levels can be measured. Diagnosis in the definitive host Necropsy The sedimentation and counting technique (SCT) is considered the gold standard (see Glossary) and the most accurate quantitative method for both species of Echinococcus [7]. The intestine is opened and incubated in physiological saline and the intestinal mucosa is scraped with a spatula. The released worms can be retrieved and counted from the sediment using a binocular microscope. Glossary Conditional dependence: two tests are conditionally dependent when the sensitivity (or specificity) of the second test depends on whether the results of the first test are positive or negative among infected (or non-infected) individuals. Latent class models: these models estimate the test sensitivity, specificity and prevalence (i.e. unknown latent classes) using observed test results. If properly structured, they can be used to estimate the true prevalence of disease or infection in the absence of a gold standard or reference test. They can also be used to develop better reference tests. Negative predictive value: this is the probability of an individual being healthy or non-infected given the observed test result. This also depends on the test characteristics and true prevalence within the population. Perfect gold standard: a test which has both a specificity and sensitivity of 1 or 100%. A perfect gold standard accurately predicts disease or infection status in every individual and in every stage of infection. In practice, a perfect gold standard is rarely available. Positive predictive value: this is the probability of an individual being diseased or infected given the observed test result. This depends not only on the test characteristics but also on the true prevalence within the population. Reference test or gold standard: the reference test is usually an imperfect gold standard. It will be the best available test to define the infection or disease status of an individual. The sensitivity and specificity of the test are usually well defined but often fall short of a perfect gold standard. Sensitivity: the proportion of true positives the test detects. A test with a sensitivity of 0.98 or 98% will be positive in 98 of every 100 truly diseased or infected individuals. Specificity: the proportion of true negatives the test detects. A test with a specificity of 0.98 or 98% will be negative in 98 of every 100 healthy or noninfected individuals. Test prevalence: the proportion of individuals in a population that test positive to a diagnostic test at a single time point. Test prevalence is only equal to true prevalence if the test used is a perfect gold standard. True prevalence: the proportion of individuals in a population which have the disease or infection at a single time point.
Corresponding author: Torgerson, P.R. ([email protected]).
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1471-4922/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2008.12.008 Available online 5 March 2009
Review The intestinal scraping technique is a less laborious method for mass screening and has a sensitivity that is 78% of that of the SCT [8,9]. Deep mucosal scrapings (a total of 15 per intestine) are made using microscope slides, and these are squashed into thin layers and examined microscopically, enabling semiquantitative estimation of the worm burden. Safety precautions must be strictly followed when using either of these diagnostic strategies [10]. Purgation Oral administration of arecoline hydrobromide to dogs results in the purgation of intestinal contents after 30– 60 min. This material can be examined for the presence of Echinococcus parasites. This technique was used during the eradication campaign in New Zealand for mass surveillance [11] and has been utilized in many epidemiological studies worldwide. The technique is nearly 100% species specific and has been used for quantitative studies. However, safety precautions during field work and parasite identification in the laboratory are essential and time consuming [10]. Arecoline can also cause serious adverse reactions in dogs. The sensitivity is poor: studies have generally assumed that the sensitivity of the technique is 65% [12]. However, a recent study indicates that it might be as low as 38% for E. granulosus and 21% for E. multilocularis [13]. Nevertheless, it is a potentially useful technique, particularly when evaluating other diagnostic procedures such as copro-antigen or copro-DNA tests, because it can prove the presence of infection. Copro-antigen enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assays (ELISAs) for antigen detection in dog faeces were initially developed independently by several groups. Initially developed for the diagnosis of E. granulosus [14,15], ELISAs for the detection of E. multilocularis subsequently became available [16,17]. Sensitivity of copro-antigen assays is generally good with moderate-to-high worm burdens (>100 worms) but less in animals with low burdens. Copro-antigens can also detect prepatent infections [14,18–20]. Although sensitivity and specificity have often been defined in selected groups of animals (e.g. experimentally infected animals), the actual performance in the field is less certain because of potential crossreactivity with antigens from Taenia species or other helminths. Furthermore, test parameters will vary with the population on which the test is used. In addition, even with high test specificity, the positive predictive value can be poor if used where the Echinococcus prevalence is low. This occurred in Cyprus in a study of the dog population during the final stages of a control programme to eliminate the parasite [21]. However, coproantigen tests remain useful procedures for population studies, provided the potential pitfalls are fully understood, because they can be performed in both living and dead animals. DNA-based tests Highly specific PCR tests have been developed for use on faeces or on eggs isolated from faeces to confirm the presence of Echinococcus spp. [22–26]. Because PCR is costly and time-consuming, it is not suitable for routine
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diagnostic or large-scale purposes. PCR-based diagnosis of intestinal infections should be implemented within a diagnostic strategy. Copro-PCR is best used for confirmatory purposes of copro-antigen-positive samples (Box 1). Alternatively, it can be used as the preferred method for identification of morphologically indistinguishable Taenia and Echinococcus eggs recovered from samples. A considerable problem with working on faeces is that to obtain a good sensitivity, large volumes are required. PCR procedures might also suffer from inhibition by faecal components, which lower diagnostic sensitivity [27]. One approach to overcome these limitations, which is only suitable for patent infections, is to undertake PCR on isolated taeniid eggs [23]. The use of the sieving-flotation method [23] in this context seems to be more sensitive at detecting taeniid eggs than other methods, such as the McMaster method [28]. A study in Kyrgyzstan has indicated that the sensitivity of egg isolation followed by PCR is 78% for E. granulosus infections and 50% for E. multilocularis infections [13]. This could seem low; however, when the length of the prepatent period and life expectancy of the parasite are accounted for then, arguably, the Box 1. Multiple tests Multiple diagnostic tests are often used on populations. This could include a screening test of high sensitivity and a confirmatory test of high specificity or parallel screening using two (or more) tests on the whole population. If tests are conditionally independent, an individual testing positive to both tests has a high probability of being a true positive (see Table I in Box 2). In humans, ultrasound relies on direct visualization of the parasitic cyst and serology relies on the immunological response to the exposure to the parasite. Consequently, these two tests can be regarded as independent. By contrast, in dogs, copro-antigen, copro-PCR and arecoline all directly detect the presence of the parasite. These tests might, therefore, have a dependence structure. In one theoretical scenario, a prevalence of E. granulosus infection is assumed to be 20% and a copro-PCR test with a sensitivity and specificity of 78% and 93%, respectively, is used. Lack of specificity might be due to coprophagia [13]. In addition, a copro-antigen test of sensitivity and specificity of 88% and 95%, respectively [70], is also used, and this is based on proved positive infections as determined with arecoline treatment or negative dogs in a nonendemic area having received antiparasitic treatment. Conditional independence of the tests would indicate a positive predictive value of 0.98 if both tests are positive. This compares to the individual positive predictive values for copro-antigen and copro-PCR of 0.81 and 0.75, respectively. When there is conditional dependence – for example, with a dependence coefficient of 0.01 between the two sensitivities and 0.02 between the two specificities – the predictive value of two positive tests would be only 0.88. In a second theoretical scenario, there could be a low prevalence of infection of 2%, perhaps after a control programme. The specificity of coproPCR would then be virtually 100% because there would be little intestinal passage of eggs owing to coprophagia. Then, conditional sensitivity of the two tests would make only a small difference to the results. In reality, expensive copro-PCR is only likely to be used as a confirmatory test. Therefore, in the first scenario, if tests are conditionally independent, confirmation by PCR indicates a test prevalence of 0.14, compared to a test prevalence of 0.16 with conditional dependence. In the second scenario, the test prevalence is 0.014 for the independent model and 0.015 for the dependent model. Paradoxically, more samples will be confirmed by PCR as positive if the tests are dependent than if the tests are independent. Thus, a good approach would be to have a screening test of high sensitivity followed by a highly dependent confirmatory test of high specificity.
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Review sensitivity for the detection of patent infections rises to 87% and 72%, respectively. Attempts to improve the sensitivity could include repeated faecal sampling and/or using PCR techniques that do not rely on egg isolation, thus detecting prepatent infections [29]. However, the dynamic of coproDNA excretion is dependent on loss of parasite stages (protoscoleces during the first days after inoculation and immature stages at the end of prepatency), whereas coproantigen concentrations are related in this infection period to parasite metabolic activity [29]. Direct detection of DNA in faeces of E. granulosus-infected dogs revealed 15 dogs positive by copro-PCR during the prepatent period of 58 dogs proven to be infected by E. granulosus, indicating a sensitivity of 26% during this phase of the infection [30]. A further important limitation of copro-PCR is that formalin-fixed faecal material is not suitable, owing to DNA degradation. However, faecal material stored in 70% ethanol or between 20 8C and 80 8C can be used [29]. Serology in definitive hosts Serology for detection of intestinal E. granulosus infections in dogs [31] and E. multilocularis infections in foxes has been investigated. However, problems with sensitivity, specificity and previous infections have prevented specific antibody detection being employed in surveillance studies [32–34]. Diagnosis in intermediate hosts Natural intermediate hosts of E. multilocularis are many small mammal species, and necropsy is the usual method of detecting infection in them. Metacestodes that contain protoscoleces can easily be identified microscopically. However, small or calcified fibrotic lesions of 0.5–5.0 mm in diameter can often be efficiently identified by PCR. In an investigation in 889 water voles (Arvicola terrestris) in a high E. multilocularis-endemic area, 277 liver lesions were detected. Of these, 108 were caused by Taenia taeniaeformis and 26 contained E. multilocularis protoscoleces, as identified by classical morphology. From the remaining small lesions, 55 were positive and 106 lesions were negative by E. multilocularis PCR [35]. For ecological studies, the proportion of protoscolex-containing animals and protoscolex numbers are important for parasite reproduction estimations in intermediate host populations. However, the total number of infected animals represents the infection pressure in a rodent habitat. There are increasing reports of AE in domestic dogs and zoo animals [36]. In these cases, diagnosis in the individual live animal is important. Imaging and serological techniques have been developed and are similar to those used in humans. However, as the dog can be both a definitive and an aberrant intermediate host, any serological test should, ideally, distinguish between these two possibilities. Alternatively, serology combined with copro-antigen or copro-DNA tests will indicate the presence or absence of intestinal infections [37]. Pigs with lesions of AE, confirmed by molecular techniques, have also been described [36]. Even though infections in pigs do not contribute to transmission, they are excellent markers for environmental contamination with eggs. Diagnosis for E. granulosus is rarely indicated in individual farm animals, but population surveys are important 166
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in obtaining baseline information about the parasite epidemiology, particularly in respect to a control programme [38]. Generally, diagnosis in sheep is at postmortem [39]. Sheep slaughtered for meat are often young and will inevitably have a lower prevalence than older animals [38–40]. Furthermore, animals likely to fetch the best price might be sent to the abattoir, and these might be animals that are better conditioned with a smaller likelihood of infection. Thus, abattoir studies, especially if they are not age-stratified, can produce substantial underestimates of the true prevalence. Ultrasound examination can be used in sheep to detect hepatic but not lung cysts. However, sensitivity is disappointing. For example, in Tunisia, mass screening of sheep by ultrasound indicated a prevalence of hepatic echinococcosis of 20% [41]. However, sensitivity is disappointing: studies in Kenya indicate that the sensitivity of ultrasound examination for detection of CE in sheep and goats is 54%, with a specificity of 94% [42]. Immunodiagnosis in sheep presents problems of sensitivity and specificity, limiting its use. Indirect haemaglutination, double diffusion and ELISA have all been attempted with little success [43]. Poor specificity is often attributed to crossreactivity with other taeniids (such as Taenia ovis and Taenia hydatigena). Recent studies using hydatid fluid antigen have reported a sensitivity and specificity of 89% and 90%, respectively, based on macroscopic lesions as the gold standard. This test gave good discrimination against ovine cysticercosis, and the specificity was improved using purified fractions of hydatid cyst fluid (S2B) but with a loss of diagnostic sensitivity [43]. Previous studies using hydatid cyst fluid, antigen B (AgB) purified from hydatid cyst fluid or protoscolex antigens have reported sensitivities ranging from 36% to 90% and specificities ranging from 65% to 96% [44–46]. Diagnosis in humans Ethics require accurate diagnoses in population studies so that the best advice regarding appropriate treatment can be given. Diagnoses are complicated in CE and AE coendemic areas, such as the Tibetan plateau and the Baltic States. The human prevalence can be as high as 5% for both CE and AE in some highly endemic areas of the Tibetan plateau [47], but in general, studies have demonstrated prevalences of <2% and often much lower [48,49], even in areas with high animal prevalences. With such low prevalences, the diagnostic specificity becomes important for accurately identifying individuals who might require treatment. For example, consider a diagnostic test that has a specificity of 98% and a sensitivity of 90% in a large survey of 10 000 people with a true prevalence of 1%. There will be 100 individuals who have hydatid disease, of which 90 will test positive. There will be a further 198 (2%) of nondiseased individuals who will also test (false) positive. In this case, the positive predictive value is 31.3% (90/288). Although the probability of having the disease has increased from 1% (pre-test) to 31.3% (post-test), the test-positive individual is still more likely to be healthy than diseased. (This problem is further illustrated in Box 2.) Highly specific tests are required, multiple tests (such as serology and ultrasound) should be used and accurate interpretation of results is vital. The negative predictive
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Box 2. The expected test results in a hypothetical human screening programme For this calculation, a hypothetical cross-sectional study of 10 000 individuals could be undertaken and echinococcosis diagnosed by ultrasound and serology, assuming the tests are conditionally independent. The specificity of ultrasound has been reported to be 0.96 in the detection of abdominal cysts [71]. To take into account pulmonary echinococcosis, the sensitivity could be assumed to be 0.91, although it could be lower. Sensitivity and specificity of recombinant antigen B (rAG138/2) have been reported at 0.83 and 0.98, respectively [72]. In addition, the sensitivity and specificity of serology is assumed to be the same if patients have hepatic or pulmonary echinococcosis, although in reality this is not likely [54,73]. Based on a true prevalence of 5%, 2% and 1% in the screened population, Table I gives the positive predictive values and expected results of the combination of test results given these assumptions regarding the diagnostic test performances. If ultrasound screening is undertaken and serology is only used as a confirmatory test, then with 5% prevalence, 387 cases would be
diagnosed as CE, of which 8 would be falsely diagnosed. There will also be 486 indicated by ultrasound to be CE but negative by serology. If these cases are further investigated or followed up, it should reveal 76 of these as true positives. This would leave 45 undetected cases in the population. If the whole population is also screened with serology regardless of ultrasound test, there would also be 192 cases that are ultrasound negative but serologically positive. Of these, 38 would be true positives. These could be offered a chest x-ray and follow-up. Seven CE cases would still be negative for both serology and ultrasound. The corresponding numbers for the prevalence of 2% and 1% can easily be found in Table I. Unlike animals, the actual diagnosis in individuals participating in a mass screening is important. However, as illustrated above, a precise diagnosis cannot be given, although the probability of infection can be estimated given the test results and estimated population prevalence. Appropriate recommendations on a case-by-case basis must then be made.
Table I. Positive predictive values in a hypothetical human screening programme of 10 000 individuals Test results Tested positive by both tests
Tested positive by ultrasound only
Tested positive by serology only
Tested negative by both tests
True prevalence Positive predictive values Number testing positive True positives Positive predictive values Number testing positive True positives Positive predictive values Number testing positive True positives Positive predictive values Number testing negative True positives
value in this case is 99.9%; thus, a test-negative individual is unlikely to have hydatid disease, further illustrating that accurate investigation of positive test individuals is important. Ultrasound is widely used and can confirm the diagnosis of abdominal echinococcosis and indicate if lesions are active. Further information on the use of ultrasound can be found in many sources (for example, Refs [49–52]). The main disadvantage of ultrasound is that it cannot normally detect pulmonary echinococcosis. In an ultrasound study based on autopsy records of asymptomatic cases of CE, a ratio of 8.3:1 of liver to lung cysts was recorded [53]. This indicates the sensitivity of ultrasound in mass screening can be as low as 89%, even assuming it has a sensitivity of 100% in detecting liver and abdominal cysts. One screening programme that also used a portable x-ray found a prevalence of pulmonary echinococcosis of 1.1% with an overall prevalence of 5.5% [54]. Therefore, although ultrasound for mass screening is generally considered to have a sensitivity of 88–98% and a specificity of 93–100% for abdominal lesions [49,55], the sensitivity for detecting all forms of echinococcosis is somewhat lower. The sensitivity and specificity to detect AE lesions might be comparable to hepatic CE as primary lesions occur in the liver. However, sensitivity could be reduced because slower developing lesions make early diagnosis more difficult and also compromise specificity. Advanced imaging techniques such as nuclear magnetic resonance or computeraided tomography can be used to confirm diagnosis in humans [51,56] for both forms of disease. Unfortunately, in many remote endemic areas, such facilities are not
5% 0.982 387 379 0.155 486 76 0.196 192 38 0.00084 8935 7
2% 0.954 159 152 0.07 454 30 0.09 174 15 0.00032 9213 3
1% 0.911 83 76 0.034 443 15 0.045 168 8 0.00016 9305 1
available. In this case, serological back-up tests might be necessary to give additional information regarding the nature of the lesion detected by ultrasound. Several serological assays have been developed for the diagnosis of human echinococcosis (reviewed in Refs [57,58]). Native crude antigens, such as hydatid cyst fluid or crude E. multilocularis metacestode material (a complex mixture of glycolipids, lipoproteins and carbohydrates), have generally proved to be sensitive for both CE and AE. However, in a variable proportion of AE or CE patients, no specific antibodies could be demonstrated with a variety of tests. This could be due to complex parasite– host interactions modulating the immune system [59,60]. Specificity is a problem with crossreactions with other helminth species [61]. Specificity was considerably increased on the genus or even species level by purification of antigens such as AgB in hydatid cyst fluid of E. granulosus or Em2 in E. multilocularis metacestode material. Various groups have developed diagnostic tests based on recombinant AgB, which produces better results than native AgB (reviewed by Refs [58,62]), and synthetic peptides mimicking defined epitopes have also been investigated. Sensitivity and specificity of tests based on AgB have largely been evaluated on known hydatid disease patients or known healthy individuals, in which sensitivity has varied between 0.45 and 0.92. Specificity is reported as between 0.71 and 1 with crossreactivity against AE, cysticercosis, and Schistosoma and Toxocara parasites [58]. The diagnostic performance of different antigens for the diagnosis of AE has enabled their use in the diagnosis of individual patients and use in seroepidemiological studies. 167
Review Reported sensitivity ranges between 0.86 and 0.97, with a specificity of 0.61 to 1.00 [63]. Furthermore, recombinant Em18 and Em16 antigens are predominantly recognized by sera from patients with active lesions of AE [57]. In addition to clinical echinococcosis, serology detects exposure to the parasite before disease development or without parasite establishment. In a pig model for AE, specific antibody reactions against E. multilocularis- or E. granulosus-derived antigens were detected one month after experimental inoculation. Interestingly, animals with multiple small lesions of approximately the resolution limit of most imaging techniques showed stronger antibody reactions compared with animals with fewer, larger lesions [64]. In Germany and Switzerland, human serological studies have been undertaken in which several subjects had a serological response against highly specific E. multilocularis antigens with undetectable or only inactive lesions [48,65]. Likewise, in China, serological prevalence of both CE and AE among children was reported to be much higher than actual disease prevalence [66]. Some work has been undertaken to develop tests for circulating antigens to diagnose echinococcosis (see, for example, Ref. [67]). Such tests have not been widely used so far. Population studies in the absence of a reference test In animals, only careful necropsy approaches a perfect gold standard. In humans, advanced imaging techniques such as nuclear magnetic resonance or computer-aided tomography can be used to confirm diagnosis [51,56]. Many investigators understand diagnostic test limitations and make adjustments to test prevalences to estimate true prevalences. However, diagnostic tests are often validated on defined populations of animals, such as dogs with proven infection, either experimentally or through necropsy studies or tests with 100% specificity, such as arecoline purgation. Negative populations might include dogs that have been recently treated with praziquantel. In humans, tests might be validated using cases confirmed by surgery or cases that have classic ultrasound images. This gives valuable information on the test performance in that particular group of animals or humans, but caution must be taken when extrapolating to populations for surveillance. Test characteristics often vary with the population investigated (Box 3). When using tests for population studies, these limitations should be considered. There are now modeling techniques that can overcome the problems of undefined or poorly defined test characteristics (Box 3). The population prevalence can be estimated even when there is little a priori information known about the infection status of any individuals in the population or when there is no available gold standard. Such Bayesian techniques were used in population studies of dogs in Spain using serology and copro-antigen tests to define the true prevalence of infection [68]. In human surveillance, this could, theoretically, be a powerful technique, particularly because ultrasound and serology are likely to be conditionally independent. However, because prevalences are typically low, it would be a challenge to find populations of significantly different prevalences to develop this model. 168
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Box 3. The Hui-Walter paradigm For most diseases, there is a lack of a perfect gold standard, yet recent statistical techniques have been developed that can evaluate diagnostic tests and estimate true prevalence in the absence of such a standard. Most of these techniques use latent class models based upon a Bayesian framework [74,75], but maximum-likelihood-based approaches can also be used. True prevalence and the other latent classes of test sensitivity and specificity can be estimated if at least two conditionally independent tests are used on two populations of a priori known different prevalences. If only one population is available, three tests must be used. Hui and Walter [76] state that any combination of several tests (R) and populations (S) can be used to estimate the true prevalence and test characteristics provided, as long as S R (2R 1 –1); that is, R = 3 tests and S = 1, population will suffice. The numbers of degrees of freedom in the data must be equal to or greater than the number of unknown parameters in the model. Adding an additional population, for example with the two-test model, adds three degrees of freedom but only one extra parameter (the prevalence of the second population). If, however, the tests have conditional dependence, then in the case of the two-population two-test model, the problem becomes nonidentifiable because there are now two additional parameters (i.e. the conditional dependence of the sensitivity and specificity of the tests). Even if further populations are added, the problem remains unidentifiable. The same is true if further tests are added that are not conditionally independent [77]. If conditional independence cannot be assumed then prior information regarding some test characteristics is required. This was the approach used in a study in Kyrgyzstan [13]. Arecoline purgation and copro-PCR were used as the two diagnostic tests and the population of dogs was age stratified (E. granulosus) or stratified by risk factors (E. multilocularis). Because the specificity of arecoline purgation was 1, at least two parameters disappear from the model (specificity of arecoline and the conditional dependence of the specificity of arecoline and copro-PCR). This then enabled the model to become identifiable. However, the HuiWalter paradigm also assumes constant specificity and sensitivity across populations of different prevalences. This cannot always be assumed because sensitivity and specificity can vary [77]. In echinococcosis, there might be variations in crossreacting helminths between different populations and, hence, different rates of crossreactivity. The additional factor that needs to be considered is not only the point estimates but also the confidence intervals or degree of certainty. This is affected by both the sample size and the prevalence. A low prevalence means there are few true positives on which to calculate the sensitivity. For example, a copro-antigen test was described as having a sensitivity of 77% on the basis of being positive in 20 of 26 purge-positive dogs [33], but the confidence intervals are 56–91%. However, specificity can often be well defined when the prevalence is low because most individuals are true negatives.
A further important limitation of using test results in epidemiological studies is risk-factor analysis. Such studies define animals or humans who might be at particular risk of echinococcosis in a population. Standard logistic regression analysis will give risk factors associated with the test results and not necessarily the disease status. Thus, in the case illustrated in which the positive predictive value is 31.3% (despite a specificity of 98%), logistic regression analysis without adjustment for true disease status could give more information about the risk factors for false-positive test reactions rather than disease transmission. Bayesian techniques are now becoming available to analyze risk factors for true disease status, rather than the test results (see, for example, Ref. [13]).
Review Concluding remarks Accurate diagnosis of echinococcosis is challenging. In animals, diagnosis is usually required at a population level. Provided that characteristics of diagnostic procedures are accounted for, meaningful results can be obtained, which can be used in epidemiological studies or for assessing progress of control programmes. In humans, there is a need for population studies and the same caveat applies to estimate population prevalences. However, in such studies, it is also essential to optimize decision making for the individual subject, and here, the lack of a perfect gold standard can make this demanding. For the individual who is diagnosed with imaging techniques but who is subsequently found not to have echinococcosis, this is not necessarily a problem because the subject might have some other space-occupying lesion that requires treatment. Furthermore, the prognosis is improving for AE with modern treatment methods [69] and, hence, early diagnosis is likely to improve the treatment outcome. In a situation in which multiple tests (such as serology and ultrasound) are used, care must be taken when tests give conflicting results because there is a possibility that the subject has echinococcosis. Such subjects should be followed up and appropriate action taken. A screening test of high sensitivity should ensure most cases are detected, but confirmation with a highly specific test is then indicated. Surveillance on human populations with serology as the single diagnostic test must be undertaken with extreme caution, if at all. Even with a serological test of high specificity, the positive predictive value will be low because of the low population prevalences.
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