Survey and Diagnostic Challenges after Transmission-Stop: Confirming Elimination of Schistosomiasis haematobium in Morocco

Clinical cases of Moroccan residents have been recorded since 2004, indicating successful interruption of transmission of S. haematobium infection at national level. The first national survey initiated in 2009 for Schistosomiasis haematobium among children born after 2004, applied diagnostic test was the HAMA-EITB, based on the Western blot technology, and molecular malacological diagnostic tools clearly confirm transmission stop. In 2015, a recent, small survey utilizing an HAI, ELISA tests and an ultrasensitive antigen test, FTCUP CAA, in a group of individual with a past history of infection. However, obviously follow-up surveys to prevent reemergency and for certification of the schistosomiasis elimination require vigilant diagnosis strategies. Here we discuss diagnosis story line in the national laboratory and challenges based on the available tools in relation to their clinical parameters (sensitivity/specificity; Sn/Sp), practicability and associated costs. When transmission stop has been achieved, survey cost and speed are likely to benefit from cost effective pooling strategies and ultrasensitive assays indicating active infection in all potential risk groups. Similarly molecular pooling strategies to monitor infections in the snail vectors.


Introduction
Schistosomiasis haematobium is a worldwide public health problem affecting 150 million persons in third-world countries. It was estimated that annual mortality due to nonfunctioning kidneys caused by infections with S. haematobium could be as high as 150,000 [1,2]. Although morbidity and mortality was significantly decreased a er introduction of programs that include large-scale drug therapy, estimates are that in 2015 at least 218 million people still require preventive treatment to restrain transmission [3]. National programs applying repetitive (annually or bi-annually) mass drug administration (MDA) approaches have been implemented in several countries in Africa [4]. e immediate impact of (repeated) chemot.herapy in case of S. haematobium infection is a decrease in the number of severe cases of urinary schistosomiasis and the concurrent reduction of the parasite load, in general this leads to overall lower endemicity. However, long term success of any MDA approach depends on additional measures as behavioral changes, improved sanitation and access to clean fresh water. As schistosomiasis is a vector-borne disease, actions to constraint or eliminate the intermediate host (i.e., snails from the genus Bulinus) are apparently mandatory as well. In MDA low endimicity established settings, new-and re-infections will likely continue when the secondary measures are not sufficient. Moreover, new cases and low worm burden may not be recognized effectively because they are not easily detected by the generally used low sensitive egg microscopy and poor specificity of serological (antibody detection) methods. In these settings the proper timing of MDA-stopping decision is difficult when evidently the required appropriate diagnostic tools for monitoring are missing. Highly sensitive diagnostic tools are therefore required to successfully proceed-to and validate the elimination of the disease [5].
Nevertheless, in several countries, morbidity has been successfully controlled and transmission thought to be interrupted as a consequence of intensified efforts. e status of schistosomiasis in areas that have reached low-transmission was reviewed not long ago [6] and recommendations offered regarding tools and strategies for monitoring, criteria to determine interruption of transmission and validation of elimination. However, these recommendations do not (yet) include the implementation of the newly developed ultrasensitive diagnostic tools [7, 8] that allow highly accurate determination of the infection status [9][10][11][12][13][14][15]. It is believed that transmission-stop has been achieved in 19 countries, and currently some already await confirmation of successful steep towards elimination. An unequivocal approach including the use of accepted diagnostic tools is crucial to demonstrate actual elimination status. e aim of our review is to present the line story of the disease in Morocco, the description of the diagnostic considerations for verification of elimination, and cost effective diagnosis strategy purposed to certify schistosmiasis elimination.

Schistosomiasis Transmission Interruption in Morocco
Morocco is one of the countries thought to progress rapidly towards elimination. Urinary schistosomiasis caused by S. haematobium and transmitted by Bulinus truncatus, was first diagnosed in 1914 in Marrakech. It became highly endemic and was given priority in public health work in the early eighties [16]. e intervention was successful, and at 2004, the interruption of transmission at national level was apparently achieved. Since 2004 no new Schistosoma infection in Morocco of a native resident has been recorded.
Five years later, an initiative to confirm interruption of transmission and first step to towards endorsement of elimination was started in 2009 with a national serological survey for Schistosomiasis haematobium among children born a er 2004. e areas selected to participate were the past schistosomiasis endemic provinces: Errachidia, Tata, El Kelaa des Sraghna, Beni Mellal, and ChtoukaAit Baha. e applied diagnostic test was the HAMA-EITB, based on Western blot technology. e test screens for the presence of antibodies against microsomals S. haematobium worm antigens with presumptive clinical sensitivity and specificity of 99%. e results demonstrated absence of new antibody positive cases for all 2382 individuals included in the survey [17]. A sometime later performed national malacological survey that simultaneous use of DraI PCR and the specific Sh110/Sm-SL nucleic acid PCR test in past endemics areas in Morocco indicated absence of S. haematobium in its specific snail vector Bulinus truncatus. Although the survey indicated the potential presence of S. bovis, results are considered good confirmation for transmission stop of human infections by S. haematobium as also indicated by the serological survey [18]. Hence, these two surveys and the fact that no new cases of urinary schistosomiasis have been reported suggested successful elimination of the disease in Morocco. en obviously follow-up routine surveys to prevent reemergency require vigilant and cost effective diagnosis strategies.

Post Elimination (Diagnostic Tests Challenges)
3.1. Antibody Detection Test. Antibody detection as recommended by WHO [19] obviously is well suited to test individuals born a er transmission stop followed by antigen or nucleic acid based testing for those testing seropositive cases [20]. However, antibody testing for travelers, immigrants and residents born before transmission stop was claimed does not seem to be a valid approach as there are no accurate antibody tests that can distinguish past from previous infection. Moreover, sensitivity as well as specificity of the current antibody assays may be an issue [21,13]. Relevantly large inconsistencies between results obtained with different antibody tests still make the general application of a single antibody test challenging [22]. Algorithms based on the use of multiple antibody tests would be needed to obtain the required high sensitivity. When targeting maintaining transmission stop, specificity seems somewhat less important as treatment of few false positives is not disastrous although surely not recommendable in view of possible drug side effects and potential drug resistance. However, to certify elimination specificity cannot be neglected. Appropriate algorithms based on parallel rather than serial testing may partly deal with specificity issues but it will relevantly increase cost. HAMA EITB is highly sensible and specific, using pooling samples, but strep test preparation required worm Schistosoma haematobium culture and high technology (the antigen must be ordered from committed international laboratories one year before). Antigen and to a lesser extend nucleic acid based testing may then become valid alternatives especially when high sensitivity and specificity can be warranted even when testing using pooling strategies. As is the case in several other countries, in Morocco the tool used for routine monitoring is still the direct examination of the urine of children with a focus on the (previous endemic) high risk areas. e detection of the presence of viable eggs in urine defines an active case [19]. Given the large migration of African refugees from endemic countries during the last decade and the real probability to miss the low-worm/egg burden cases, it is prudent to improve the epidemiological tools for children serological survey in high risk areas.

Antigen Detection Test.
In 2017, small study amongst 37 adults with a history of past infection (before 2004) indicated 2 individuals still carrying residual but low worm burden Schistosoma infections; the actual involved species was not determined [13]. e applied diagnostic tool detected a wormspecific carbohydrate which is discharged by alive worms into the human blood circulation, the circulating anodic antigen (CAA). is bio-molecule is detectable in blood and/ or urine and indicates an active ongoing infection, but does not always imply egg production. e CAA test is a highly specific and the most sensitive assay capable to detect a single worm Schistosoma infection including non-fecund or singlesex worms. It is a genus specific test that will identify active infections with any of the know Schistosoma species, including bovis, curassoni and intercalatum [13]. e identification of the two individuals with residual infection indicates the importance of the need of proper and highly sensitive diagnostic tools to obtain accurate information regarding elimination status. e infected individuals were non-symptomatic and likely carry a low worm burden as indicated by the measured level of CAA, with worms probably incapable of egg production. However, it seems clear that to maintain the currently alleged elimination of urogential schistosomiasis in Morocco and to prevent reemergency of infection either through unexpected species, residual low worm burden infections in adult residents, or through infected immigrants and travelers, a highly focused strategy and survey protocol is needed.

Selecting Suitable Diagnotic Tools for Current Surveys
Surveys may include parasitological methods, serology (antibody detection) and any newly available test as e.g. assays detecting worm antigen in bodily fluids. e surveys should also include snail surveys as presence of the vector by itself does provide an important pulpit for re-emergence of the disease. Besides Sn and Sp issues of the applied tests, costs issues play a role and in part determine the scale at which the surveys can be performed. Possibilities to implement accurate and cost-effective pooling strategies may thus be of importance. For the surveys three types of assays are considered: (i) assays detecting active Schistosoma infection for individuals born before 2004 and immigrants; (ii) assays detecting exposure to S. haematobium (Sh) and S. bovis (Sb) for individuals born a er 2004; (iii) assays detecting snails infected with miracidia. In Table 1 a number of tests are listed that were previously used, and/or are considered for future use in national surveys to detect Schistosoma infection. We note that not all tests are (always) commercially available and price-ranges can be quite variable, but obviously when surveys are part of scientific studies (including the evaluation of laboratory based 'researchonly' tools) or vice versa, noncommercial diagnostics should not be excluded. High cost [20,33] of DraI reverse primer and Sh73 direct primer (73d). e sensitivity of S. haematobiumdetection was 1 pg/μl, whereas S. bovis detection was 10 pg. It can be assumed that detection of snails infected with S. haematobium will be accomplished from very early prepotency. Such assays required further validation using larger numbers of field snails in Morocco [41,18].

Human Survey.
e process of confirming the interruption of transmission and possible certification of elimination is based on human and snails survey each five years since 2009, with the third survey coming up in 2019. e national survey of Schistosomiasis haematobium is largely focused on children born a er transmission-stop 2004. Surveys will be performed in settings in past endemics provinces and in areas were imported African cases were registered during the last years. As diagnostic test, besides individual urine parasitological, the UCP-LF antigen assay for CAA detection also in individual sera and urines (Figure 1).

Malacological Survey. Belkacemi et al. reported that
March is the month of reproduction of snails in Morocco, and juvenile snails become infective in the beginning of September [48,49].Transmission period is greatly associated with population movement generally important in summer August-Octobre (holiday, travel, swim, irrigation) [49,50] PCR of snail pools has been shown to be practical for large-scale monitoring of S. haematobium transmission, however there is a need to improve the cost-effectiveness of the test by development of a method similar to LAMP amplification that is cheaper and more field friendly [40]. e malacological treatment may be done annually in each breeding sites showing Bulinus in the last three years, because, treatment can le the juvenile snails, and the life age of Bulinus is estimated to two years [51,52].
for dried blood samples [42], serum, plasma and urine [11][12][13]. e test includes a sample preparation step which may make the assay in its current format somewhat less field-friendly as this step requires some basic laboratory equipment. However, the sample preparation step makes the assay highly flexible towards sample input (matrix type and volume) allowing improved sensitivity to the level that single-worm, single -sex and low fecundity infections are detectable. is flexibility towards sample amount also makes the assay well suited for cost-effective pooling strategies [43]. (ii) Assays detecting exposure to S. haematobium and S.
bovis for individuals born a er 2004: In China where S. japonicum (Sj) has been largely eliminated as a public health problem in through implementation of a series of different measures [44]. Detection of seropositive individuals is applied to identify potential active infections and transmission. However, a study by van Dam et al. indicated that the applied antibody test lacks specificity [21]. HAMA EITB highly specific and sensible but could be expensive. A generic rapid antibody detection platform utilizing UCP-LF has been developed for testing with various biological matrices (blood-based, urine and saliva) [7-46], but still require evaluation and validation for potential application in pooling approaches. e antibody assays do not need a sample preparation step and are usable for point-of-care (near patient) use and if needed can be performed non-invasively using urine samples [47]. (iii) Assays detecting snails infected with miracidia.
Malacological studies utilizing nucleic acid (NA) amplification to detect miracidia NA are also well suited for pooling strategies as the technology also requires a sample purification step which is quite flexible towards sample volume. Simultaneous use of DraI PCR, and Sh110 SmSl PCR, as used in our first study, take more time and reactive [18]. More recently, Abassi et al. develop a simple and more sensitive PCR assay that enables direct discrimination of S. haematobium from related animal schistosomes, by the primer combination

Conclusion
Schistosomiasis antigen detection test and malacological molecular test are suitable for human and snail survey in post elimination phases. Nevertheless to consolidate the approach survey, temporary drying of irrigation canals, sanitary education, improvement of Schistosomiasis laboratories, improvement of basic formation and training programs (hours number) of medical parasitology for students in institute of technical health even if some parasitic disease are eliminated, improvement of scientific technology, survey of imported cases and monitoring of risk map may be useful to maintain elimination, prevent reemergence of the disease and for certification of the schistosomiasis elimination.