Schistosomiasis is a neglected infection affecting millions of people, mostly living in sub-Saharan Africa. Morbidity and mortality due to chronic infection are relevant, although schistosomiasis is often clinically silent. Different diagnostic tests have been implemented in order to improve screening and diagnosis, that traditionally rely on parasitological tests with low sensitivity. Aim of this study was to evaluate the accuracy of different tests for the screening of schistosomiasis in African migrants, in a non endemic setting. A retrospective study was conducted on 373 patients screened at the Centre for Tropical Diseases (CTD) in Negrar, Verona, Italy. Biological samples were tested with: stool/urine microscopy, Circulating Cathodic Antigen (CCA) dipstick test, ELISA, Western blot, immune-chromatographic test (ICT). Test accuracy and predictive values of the immunological tests were assessed primarily on the basis of the results of microscopy (primary reference standard): ICT and WB resulted the test with highest sensitivity (94% and 92%, respectively), with a high NPV (98%). CCA showed the highest specificity (93%), but low sensitivity (48%). The analysis was conducted also using a composite reference standard, CRS (patients classified as infected in case of positive microscopy and/or at least 2 concordant positive immunological tests) and Latent Class Analysis (LCA). The latter two models demonstrated excellent agreement (Cohen’s kappa: 0. 92) for the classification of the results. In fact, they both confirmed ICT as the test with the highest sensitivity (96%) and NPV (97%), moreover PPV was reasonably good (78% and 72% according to CRS and LCA, respectively). ELISA resulted the most specific immunological test (over 99%). The ICT appears to be a suitable screening test, even when used alone. The rapid test ICT was the most sensitive test, with the potential of being used as a single screening test for African migrants.
Schistosomiasis is a fluke worm infection affecting at least 78 countries and more than 200 million people worldwide, and causing an estimated burden of 3. 3 million disability-adjusted life-years (DALYs) [1,2]. Ninety-three percent of the global cases occur in sub-Saharan Africa, mostly caused by the species Schistosoma mansoni and S. haematobium. In this area, approximately 300,000 deaths due to schistosomiasis are estimated annually [3–6]. A huge wave of migrants, in particular asylum seekers, has reached Italy from sub-Saharan Africa in the past few years [7]. The health screening of newly arrived migrants is usually limited to detect potentially transmissible diseases such as tuberculosis and scabies. Schistosomiasis, often clinically silent, is not routinely screened. Given the severe complications related to both S. mansoni and S. haematobium [8–12], and the availability of an effective and relatively inexpensive treatment [1,13,14], adequate protocols for screening and treatment of migrants are required. Further, the recent outbreak of urinary schistosomiasis in Corsica [15], originated by a West African strain [16,17], is a useful reminder that local transmission of schistosomiasis in Europe is still possible [18,19]. The estimated prevalence of schistosomiasis in African migrants, reported from a scarce European literature, ranged from 9–15% if based on use of microscopy [20,21] to 5. 8–24. 7% if based on ELISA serology [22,23]. Clearly, all data obtained from reference centers are to be taken with caution as they may not be representative of the general population. These prevalence estimates are based on relatively insensitive tests, which under represent infections with low parasite loads. Traditionally, the gold standard for the diagnosis of schistosomiasis has been considered to be the urine and stool microscopy of several specimens [24]. Microscopy is 100% specific, but the sensitivity (40–60%) varies with the intensity of infection, the number of specimens collected, and the circadian and day-to-day variation of egg counts in stool and/or urine [25–27]. The diagnosis of schistosomiasis by detection of specific antibodies (with tests generally based on crude antigens of S. mansoni) is more sensitive than microscopy, particularly in light infections [26,28]. However, commercial serologic tests for schistosomiasis have a sub-optimal sensitivity too, in particular for S. haematobium infections (ranging from 21. 4% to 71. 4%) [29]. The combinations of two or more serologic tests, markedly increased the sensitivity of serology to almost 80% [29,30]. Other diagnostic techniques have been implemented more recently [24,31,32]. A Western Blot (WB) containing S. haematobium and S. mansoni soluble antigens was used on a limited number of subjects exposed to the recent outbreak of urinary schistosomiasis in Corsica, where a hybrid form between human S. haematobium and animal S. bovis was identified [18,33]. However, the accuracy of this test has never been formally evaluated. Previously, an immunoblot produced by the same manufacturer, using S. mansoni antigens only, had showed for this species a good accuracy (sensitivity 89. 5% and specificity 100%) [34,35]. The urine Circulating Cathodic Antigen (CCA) is a rapid test, based on the identification in the urine of the genus-specific proteoglycan antigen of the schistosomal gut epithelium, regurgitated by the live adult worm only [36]. The CCA test has some limitations in identifying S. haematobium infection. In a Cochrane meta-analysis of studies conducted in endemic areas, the CCA test, compared to microscopy, showed an average sensitivity and specificity of 39% and 78%, respectively, for S. haematobium infection, and of 89% and 55%, respectively, for S. mansoni. This test has been utilized only recently with immigrants in Europe [37,38]. A rapid diagnostic test (RDT) incorporating antigens of adult S. mansoni cercarial transformation fluid for detection of antibodies in blood showed a sensitivity and specificity of 100% and 39. 5%, respectively, in a schistosome-endemic area [39]. However, the reference standard used in that study was a fecal examination using Kato-Katz concentration technique, admittedly less sensitive than the index test. No studies have been carried out in non-endemic countries. The main purpose of our study was to identify the more accurate screening strategy for schistosomiasis in a non endemic area. The target condition was schistosomiasis (caused by S. mansoni, S. haematobium, or both) rather than the infection by either species. The rationale is that, with the exception of microscopy (and potentially PCR, that was not targeted by this study), the other techniques are not able to discriminate between the two species. Furthermore, the treatment of both infections is with the same drug praziquantel. The main objective was to assess the accuracy of a series of diagnostic tests for the screening of schistosomiasis, including: a) the new, commercially available RDT (Schistosoma ICT IgG-IgM), and the Western Blot (Schisto II Western Blot IgG) (LD-BIO Diagnostics, Lyon France), both aimed at detecting S. mansoni and S. haematobium antibodies; the former test has been made commercially available recently (October 2015) and is based on the principle of the homogeneous sandwich (immunological reaction of 2 identical antigenic epitopes with the two binding sites of a bivalent antibody). b) the tests routinely used at CTD, namely: the circulating cathodic antigen (CCA) urine dipstick test for S. mansoni (NADAL CCA Bilharzia test, nal von minden, Germany), an enzyme-linked immunosorbent assay (ELISA Bordier Affinity Products, Crissier, Switzerland), and the microscopic examination of stool and urine.
Ethical clearance from Comitato Etico Provinciale di Verona e Rovigo: protocol n 33909 of July 13th, 2016. At the Centre for Tropical Diseases, all subjects submitted to any serological exam are asked to sign an informed consent for the anonymous storage of a serum specimen for any future research purpose. Parents’ or legal guardians’ consent is obtained for individuals of less than 18 years of age. The assessment of accuracy of all tests for schistosomiasis is hampered by the lack of a gold standard. In particular, microscopy (of stools and urine) is virtually 100% specific but lacks sensitivity [25–27]. On the contrary, serologic tests are known to be more sensitive but may provide false-positive results [24,29,30]. Therefore, the test accuracy was evaluated using three different standards: From one to three urine samples were obtained (from 10 a. m. to 12 a. m) over consecutive days for each patient. The urine were subjected to CCA dipstick test and to filtration method for S. haematobium egg count on the day of the sample collection [36,43]. Up to 10 ml of venous blood was collected from each patient in silica-coated tubes without anticoagulant. The serum was separated by centrifugation at 3000 rpm for 5 min. Bordier ELISA was executed on 10 μl of serum, and remaining aliquots were stored at -80°C. Both Schisto II Western Blot IgG and Schistosoma ICT IgG-IgM were performed using the latter serum. Cutoffs for each test were pre-determined prior to testing.
The proportion of positive microscopic results in the population under study was 65/373 (17. 4%). In particular, S. mansoni eggs were found in the stools of 32/373 subjects (8. 6%) and S. haematobium eggs in the urine of 40/373 subjects (10. 7%). Seven subjects (1. 9%) had both the infections. The results of the 4 index tests according to microscopy are reported in Table 1. Patients were classified as microscopically positive if ova of at least one of the two species were identified in the stools or urine. ICT and WB presented the highest sensitivity (94% and 92%, respectively) and negative predictive values (NPV): 98%, both tests. CCA demonstrated a low sensitivity (48%), while its specificity was the highest (93%). Considering S. mansoni only, CCA sensitivity increased to 72% (23/32), while that of the other tests resulted similar, in particular: ELISA, 84% (27/32); WB, 91% (23/32); and ICT, 94% (29/32). Considering samples with positive microscopy for S. haematobium and negative for S. mansoni, CCA sensitivity dropped to 24% (8/33), while that of the other tests, again, resulted similar, in particular: ELISA, 79% (26/33); WB, 94% (31/333); and ICT, 94% (31/33). We also assessed if the presence of haematuria significantly influenced the proportion of CCA positivity. This data was available for about half of the study subject (185/373 or 49. 6%). Considering subjects who resulted microscopically negative to both species, CCA resulted positive in 6/43 subjects with haematuria (14%, CI 5–28) and in 9/73 subjects without haematuria (11%, CI 5–20). The proportion of positive results according to the CRS was 38. 6% (144/373). According to LCA modeling, the proportion of subjects classified in Class 1 was 35. 6%. The results are detailed in Table 2. ICT confirmed the highest sensitivity (96% for both models), followed by WB. CCA was again the test with the lowest sensitivity, that resulted very low (29%, both models). On the other hand, although the specificity of CCA was confirmed to be higher than 90%, both CRF and LCA identified ELISA as the indirect test with the highest specificity, being over 99%. The accuracy and predictive values, according to the three reference standards used, is summarized in Fig 2. Subjects with at least 2 concordant positive results of the index tests OR with a positive microscopy (irrespective of the other results) had 91. 1% probability of being classified as cases by LCA, while all the others (negative microscopy and <2 positive index tests) had 99. 5% probability of being classified as non-cases by LCA. The concordance between the two methods of classification was excellent (Cohen’s kappa = 0. 92, indicating near-perfect agreement). Assuming using ICT as a screening test (then with a second, confirmatory test in case of ICT positivity), we assessed, using the LCA model, the positive and negative predictive values of a combination of a positive ICT (the test with the highest sensitivity) with a positive or a negative second test, respectively. The results are reported in Table 3.
Schistosomiasis prevalence was very important in the study population, composed for the vast majority of asylum seekers. Even when considering microscopy only, that is not sufficiently sensitive, the proportion of positives was higher than 17%. According to CRS and LCA, this proportion was very similar, 38. 6% and 35. 6%, respectively. The urinary, rapid antigen test CCA was unsatisfactory: its sensitivity was poor, and this, with any reference standard used, makes it inadequate for screening purpose. The higher sensitivity found by other studies [37] probably reflects higher, average parasitic loads and a different reference standard based on microscopy only. Therefore, CCA is possibly a useful test (for S. mansoni) when the goal is to identify infections in an endemic country, but, according to our results, its sensitivity in non-endemic settings should be better assessed, before considering it as a potential screening tool, considering that the goal is to find and treat any infection, including chronic infections with a low egg output. WB showed an excellent combination of sensitivity and specificity, although the latter was not as high as one might expect by this technique. According to the manufacturer, a single band is sufficient to define a positive result. It is possible that a more conservative definition (i. e. requiring at least two positive bands) would bring the test specificity closer to 100%, though, expectedly, at the expense of some loss of sensitivity. We plan to investigate this with the next, longitudinal study (see below). ELISA was the test providing the most surprising results, as it was found to be virtually 100% specific according to both CRS and LCA. This result was not expected, as serologic tests in general are held to give a variable proportion of false positive results, due to cross-reaction with other pathogens and to other reasons [24]. We tried to figure out if a possible selection bias could explain this unexpected result, but we couldn’t find any, although the retrospective design of the study suggests that the results should be interpreted with some caution. According to both CRS and LCA, the sensitivity of microscopy was poor, thus confirming that finding schistosome eggs in the stools or urine is diagnostic, but their absence does not by any mean rule out the infection. All the main results, both of the infection prevalence in the study population and of the accuracy of the different tests, were very similar between the two models, indicating a satisfactory robustness of the (independent) methods used. The agreement between the two models in classifying the study subjects was nearly perfect (Cohen’s kappa test = 0. 92). Should a screening program be planned, it should rely on a sensitive approach, that minimizes the risk of leaving infected subjects without the necessary treatment. Regardless the reference standard used, the ICT resulted the most sensitive test, with the potential of being used as a single screening test (NPV >97% according to the primary and composite reference standard, and to LCA). The PPV of ICT was low when using the primary reference standard (reflecting the low specificity when a poorly sensitive test is used as reference), but reasonably high according to both the composite reference standard and the LCA, thus probably justifying a treatment, in a screening context, even without a confirmation test. Would a second test necessary/useful? The answer to this question depend on the context. Newly arrived immigrants/asylum seekers are often a very mobile group, and it is crucial to be able to screen (and treat the positives) at the first visit. The ICT appears to be sufficiently accurate for this purpose. On the other hand, in a clinical context, a confirmatory test would be obviously indicated. A positive microscopy would be the only test providing the certainty of infection, but a negative result would by no mean rule out the infection (Table 3). ELISA is a good confirmer but a weak excluder, too. (Table 3). Only the combination of more, concordantly negative tests would safely exclude the infection in a subject with a positive ICT. In summary, ICT is the ideal test for screening purpose. It is a simple test, not even requiring a laboratory or any special equipment, can be read in a few minutes and is the only test that virtually rules out the infection, if negative. This test would be even more suitable for screening purpose if validated for use on whole blood obtained through a finger prick. Unfortunately the producer has not validated this procedure. In a screening context, this test can be used alone as a tool to decide the treatment, the only reasonable alternative being, in a high prevalence population such as our study population, a presumptive treatment of all immigrants coming from high prevalence countries, considering the potential severity of the chronic infection and the relatively harmless and highly effective treatment available (praziquantel). The cost-effectiveness of either approach was beyond the scope of this research and should be planned as a further study. The new, rapid diagnostic test ICT is a suitable screening tool for schistosomiasis. A positive result should ideally be confirmed by a second test. ELISA and of course microscopy are the best confirmers of a positive ICT. Nevertheless, neither second test, if negative, is sufficient to rule out the infection. In a screening context, the ICT should probably used alone as a decision tool about treating or not the infection, the only practical alternative being a presumptive treatment of the population at risk.
Schistosomiasis is probably the most important of the neglected tropical diseases (NTD) caused by helminthes (worms). It is acquired bathing in freshwater in endemic areas. The life cycle is complex and involves freshwater snails. Schistosomiasis, caused by Schistosoma mansoni, S. haematobium and less frequently by other species, affects more than 200 million people, mostly in Africa, and may chronically cause irreversible damage of the liver (S. mansoni) or of the kidneys and the urinary tract, including cancer of the bladder (S. haematobium). As in chronic infections eggs of both species are often missed by microscopy of faeces and urine, with this retrospective study we evaluate the accuracy of different, alternative diagnostic tests, for the screening of schistosomiasis in African migrants and asylum seekers, of whom many thousands reach the Italian coast every year proceding from the most endemic areas. The most interesting finding of our study is that a rapid diagnostic test for antibody detection in blood, easy to use as a point-of-care tool, resulted the most sensitive of the five tests evaluated, and thus is very promising as a screening tool even when used without any additional test.
schistosoma
invertebrates
schistosoma mansoni
medicine and health sciences
body fluids
enzyme-linked immunoassays
pathology and laboratory medicine
helminths
tropical diseases
parasitic diseases
animals
urine
neglected tropical diseases
immunologic techniques
research and analysis methods
serology
schistosoma haematobium
immunoassays
helminth infections
schistosomiasis
diagnostic medicine
anatomy
physiology
biology and life sciences
organisms
Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis