**2. New diagnostic tools in both community and institutional settings**

The laboratory investigation of *Schistosoma* infection consists of different techniques, including parasitological, immunological, and molecular biology methods [17-19]. Frequently, diagnos‐ tic approaches are also applied on the monitoring of drug response. In addition, the assessment of morbidity levels can be achieved by using image tests and biochemical markers [20-22]. However, the diagnosis of active *Schistosoma* infection and the monitoring of therapy response as well as the determination of morbidity levels are distinctively assessed at community and institutional settings (Figure 1). Furthermore, in community settings, conventional or investi‐ gational tools aim to assess the efficiency of national control programs in the morbidity control and/or elimination of transmission by measuring the prevalence and intensity of infection in intermediary and definitive hosts [23-25]. In contrast, in institutional settings, diagnostic approaches aim to improve clinical management of individual cases.

Traditionally, egg detection by microscopy is the major criteria for active *Schistosoma* infection [24, 26]. Egg excretion can be detected by parasitological methods such as urine filtration and centrifugation methods in the case of *S. haematobium*. Since *S. japonicum*, *S. mansoni*, *S. mekon‐ gi*, and *S. intercalatum* eggs are shed in the feces, egg patent infection is detected in fecal samples by parasitological methods such as Kato-Katz test (K-K). The principal characteristics of K-K are as follows: an easy-to-do technique, low cost, reliability, and accurate identification of eggs in the case of *Schistosoma* species. Also, parasitological methods are quantitative. As a result, parasite load can be estimated. Egg counts correlate with the intensity of being <100 eggs per gram (epg), >100-399 epg, and >400 epg designated as light, moderate, and severe infection, respectively, according to WHO guidelines. Furthermore, the assessment of morbidity levels can also be roughly determined. Based on findings in high endemic areas, the elevated number of eggs was associated with severe forms of disease. Both urine filtration and Kato-Katz test have been applied for diagnosis and monitoring therapy response and used in field studies in areas of transmission as well as in institutional settings. Although Kato-Katz are affordable and suitable for low-income areas with individuals presenting with heavy to moderate infections, *Schistosoma* infection diagnosis can be quite tricky to detect in individuals with acute schistosomiasis or light infection living in nonendemic and low-endemic areas when based

**Figure 1.** Schistosomiasis flowchart for clinical management in community and institutional settings. Conventional and new tools to diagnosis, determination of response to therapy, and morbidity assessment are indicated under com‐ munity and institutional settings in hierarchic order. Bellow conventional tests, new tools were depicted according to the strength of literature evidence (red boxes). Approaches still under investigation and/or diagnostic platforms that show debatable results are inside the gray boxes. PM: parasitological method; US: ultrasonography; PCR: polymerase chain reaction; RDT: rapid test (POC-CCA /POC-CAA); FOB: fecal occult blood; CT: computed tomography; MRI: magnetic resonance imaging.

solely on microscopy [27]. Lack of egg shedding, one gender-induced infection, and daily variability are some of the causes that directly interfere with the sensitivity of microscopy, thus compromising the detection of *Schistosoma* infection and resulting in the underestimation of "real prevalence" [15, 28]. Moreover, the erratic elimination of *Schistosoma* eggs makes the determination of therapy response uncertain. In addition, some patients may present with severe forms of disease such as neuroschistosomiasis or genital schistosomiasis without any egg excretion detectable [13]. Strategies to overcome the lack of sensitivity of urine filtration and Kato-Katz test include testing replicate samples of urine or stool samples and/or aug‐ menting the number of Kato-Katz slides/sample [29].

Other parasitological methods such as sedimentation, centrifugation, flotation techniques, and miracidium hatching were developed and had improved the diagnosis of light infections by increasing sensitivity [26, 32]. In institutional settings, tissue biopsy such as rectal snips and liver biopsy are largely used to diagnose active infection in non-egg excretors despite its invasiveness [31]. Eventually, surgical specimens reveal previously undiagnosed schistoso‐ miasis. Except for rectal snips, histological examination is not quantitative, lack of information on parasite burden does not preclude clinical assistance.

Albeit the availability of diverse parasitological methods and tissue biopsies as alternatives to the reference test (Kato-Katz), nonparasitological methods were also developed to overcome microscopy false-negative results. This is the case of immunological tests, which have become more useful for showing active infections in recently exposed individuals, such as travelers or chronically infected immigrants residing in nonendemic areas [32]. In areas of transmission, immunodiagnosis is a suitable tool for surveillance in low endemic areas [29]. Several immu‐ nodiagnostic tests were developed, but currently the ELISA-based assays using egg antigen, cercarial, or adult worm antigens have been extensively used [33]. In addition, recombinant proteins and peptides have been potential targets [34-36]. Despite its infrequent use in National Programs for Schistosomiasis Control, serology is a potent auxiliary diagnostic approach that permits the diagnosis of non-egg excretors. However, the presence of active infection may be undermined by persistent reactivity despite successful treatment [13, 29]. Although immu‐ noreactivity does not correlate with the intensity of infection, data have demonstrated that isotypic immunoresponse may reflect morbidity levels [37, 38].

Moreover, rapid tests (RDT) for the detection of *Schistosoma* antigens like circulating cathodic (CCA) and anodic (CAA) antigens and DNA detection assays have proven to be an advanced and feasible strategy for diagnosing *Schistosoma* infection despite the absence of their use as routine diagnostic approaches [13, 39]. See detailed comments in Table 1. During active infection, gut-produced *Schistosoma* glycoproteins - POC-CCA and POC-CAA - are detectable in the blood, urine, and stool. At individual level, results revealed that both CAA and CCA ELISA-based assays can be quite sensitive to detect active infection early after exposure in travelers even in the cases of light infections. Also, the tests allow a quantitative assessment of antigen levels, which correlates with the intensity of infection [40]. Point-of-care platforms (POC) have been applied to estimate infection prevalence with high accuracy in field studies in high and moderate endemic areas [41, 42]. Although research groups claim that CCA and CAA might be a suitable substitute for Kato-Katz test, its performance is still debatable in low endemic areas [43, 44]. RDT for hematuria (urogenital schistosomiasis), fecal occult blood (FOB), and calprotectin detection (entero-schistosomiasis) are also point-of-care approaches, which have been shown to have fair association with egg-patent infections with dual use as diagnostic tools and markers of morbidity [25, 45]. Although strong evidences support the usage of hematuria detection by RDTs, larger studies are still necessary to establish the usefulness of FOB and/or calprotectin detection in cases of light infection commonly found in low endemic areas.
