Ultrasound and intestinal lesions in Schistosoma mansoni infection: A case-control pilot study outside endemic areas

Background Infection with Schistosoma mansoni is a major cause of morbidity and mortality in endemic areas, and is increasingly diagnosed in migrants and travellers outside transmission areas. Markers for the assessment of morbidity and impact of control programs in endemic areas and for the clinical management of patients in the clinical setting are scant, especially for intestinal involvement. Ultrasonography is well established to evaluate hepatosplenic pathology; on the contrary, ultrasound evaluation of intestinal schistosomiasis is virtually unexplored. In this pilot study, we aimed to describe and evaluate the accuracy of unenhanced intestinal ultrasound for morbidity due to intestinal S. mansoni infection. Methodology/Principal findings We performed a blind case-control study of unenhanced intestinal ultrasound on 107 adults accessing the outpatient clinic of our Centre for Tropical Diseases between January-July 2018 as part of a screening for tropical diseases in migrants and travellers returning from endemic areas. Other clinical and laboratory data were obtained routine examination reports. We could not find any overtly pathological thickness of the gut wall in the sigma, proximal ascending colon, and terminal ileum, in patients with S. mansoni infection (n = 17), S. haematobium infection (n = 7), positive anti-Schistosoma serology (n = 31), and uninfected individuals (n = 52), with no difference among groups as assessed by ANOVA. No polyps or other intestinal abnormalities were visualized. There was no significant change in gut wall thickness one month after treatment with praziquantel in patients with S. mansoni infection (n = 11). Conclusions/Significance Our preliminary results suggest that intestinal ultrasound might not be a sensitive tool for detecting minor intestinal morbidity due to schistosomiasis. Further studies in a hospital setting comparing colonoscopy and ultrasonography may be envisaged; in endemic areas, further studies are needed to describe and assess the usefulness of intestinal ultrasound in patients stratified by infection intensity and compared with markers such as calprotectin and fecal occult blood.


Methodology/Principal findings
We performed a blind case-control study of unenhanced intestinal ultrasound on 107 adults accessing the outpatient clinic of our Centre for Tropical Diseases between January-July 2018 as part of a screening for tropical diseases in migrants and travellers returning from endemic areas. Other clinical and laboratory data were obtained routine examination reports. We could not find any overtly pathological thickness of the gut wall in the sigma, proximal ascending colon, and terminal ileum, in patients with S. mansoni infection (n = 17), S. haematobium infection (n = 7), positive anti-Schistosoma serology (n = 31), and uninfected individuals (n = 52), with no difference among groups as assessed by ANOVA. No polyps or other intestinal abnormalities were visualized. There was no significant change in gut wall thickness one month after treatment with praziquantel in patients with S. mansoni infection (n = 11).

Conclusions/Significance
Our preliminary results suggest that intestinal ultrasound might not be a sensitive tool for detecting minor intestinal morbidity due to schistosomiasis. Further studies in a hospital setting comparing colonoscopy and ultrasonography may be envisaged; in endemic areas, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Introduction
Schistosomiasis is a parasitic infection caused by trematodes of the genus Schistosoma, acquired upon contact with fresh water bodies contaminated with the larval stage of the parasite. It is estimated that more than 200 million people are infected worldwide by schistosomes causing either the urinary or the intestinal form of the disease, resulting in over 200 thousand deaths every year, mostly in Sub-Saharan Africa [1,2]. Imported schistosomiasis in migrants and travellers returning from endemic areas has been defined, in receiving countries such as Italy, as a "hidden epidemic" [3].
S. mansoni, the main causative agent of intestinal and hepatosplenic schistosomiasis, is estimated to infect over 50 million people in endemic areas [4]. Pathology is caused by eggs released by female parasites residing in the venous plexus draining the large intestine, which are trapped in the bowel wall and the liver [5]. In recent years, there has been considerable effort by the international community toward the control of the infection with mass drug administration of praziquantel [1]. The aim of this strategy is the decrease in morbidity due to schistosomiasis [6]. However, morbidity markers for assessment of the infection and postinterventions evaluation are scant [7]. Also at an individual patient's level, a comprehensive evaluation of morbidity is difficult due to the variability of schistosomiasis-related clinical manifestations and lack of specific symptoms.
Morbidity and mortality due to advanced hepatosplenic involvement resulting in liver fibrosis and portal hypertension are well recognized and described [4,8]. On the contrary, morbidity due to involvement of the bowel, which is affected by the inflammatory response to eggs passing through and entrapped in the mucosa and sub-mucosa, has proven more difficult to quantify. This is partly due to the variability and lack of specificity of symptoms, which depend on the extent of inflammation and range from vague abdominal pain to bloody diarrhoea. Based on questionnaires data, diarrhoea with or without blood has been estimated to occur in over 5 million individuals infected with S. mansoni in sub-Saharan Africa, but this estimate is burdened by high uncertainty [4]. The clinical diagnostic gold standard for intestinal morbidity is colonoscopy, but this has virtually no applicability for disease burden evaluation at population level and is also poorly accepted and repeatable in asymptomatic or paucisymptomatic individuals in the clinical setting. Faecal calprotectin and faecal occult blood, unspecific markers of bowel inflammation, have been recently shown to correlate with parasite egg excretion and therefore suggested to be of use for monitoring in control programs [9]. Ultrasonography (US) is a particularly appealing tool in the field of infectious diseases because it is non-invasive, devoid of radiations, repeatable, and can be performed at the bedside of the patient with the use of portable machines [10]. Even in case of unspecific findings, the visualization of the affected organ, complementary to what provided by laboratory tests, provides useful information on the type and extent of pathology. In S. mansoni infection, US is a well-established technique to evaluate hepatosplenic pathology [10,11]. A standardized protocol for the evaluation and grading of hepatosplenic schistosomiasis using US was issued by the WHO in 2000 [12] and recently evaluated for usability and usefulness [6]. On the contrary, reports on the use of US in the evaluation of intestinal schistosomiasis are extremely scant [11,13], and there is no US report on the evolution of the lesions, when visualized, after treatment, either in endemic areas or in case of no risk of reinfection.
There is a need to find low-cost, reliable, and repeatable markers of bowel morbidity that can aid both the assessment of morbidity and impact of control programs in endemic areas and the clinical evaluation and follow-up of individual patients in the clinical setting. The primary objective of this study was to describe and evaluate the accuracy of unenhanced intestinal US signs for the diagnosis and morbidity assessment of intestinal S. mansoni infection. The secondary objective was to evaluate the evolution over time of unenhanced intestinal US signs in patients with S. mansoni infection.

Ethics statement
The study was approved by the Ethics Committee for Clinical Experimentation of Verona and Rovigo Provinces, Italy (protocol n. 63537). All adult participants provided written informed consent; children were excluded from participation.

Study design and participants
This is a case-control, cross-sectional pilot study followed by a longitudinal study of unenhanced intestinal US (Fig 1). The study was carried out on a convenience sample of asymptomatic/pauci-symptomatic individuals accessing the outpatient clinic of the Centre tor Tropical Diseases of the IRCCS Sacro Cuore-Don Calabria Hospital of Negrar, Verona, Italy, between January 1 st and July 1 st 2018 as part of a screening for tropical diseases in migrants and travellers returning from endemic areas. Inclusion criteria for the case-control study were: epidemiological risk for S. mansoni infection, age � 18 years, and no documented evidence of receiving treatment with praziquantel in the previous 6 months. This period was decided based on the known behaviour on US of bladder lesions due to S. haematobium, which resolve completely by 6 months post-treatment (in the absence of neoplastic transformation) but could be still detectable within this time interval even in the absence of active infection. Exclusion criteria were advanced pregnancy that could limit the exploration of the abdomen by US, and known acute (e.g. diverticulitis) or chronic inflammatory bowel diseases (e.g. Crohn disease or ulcerative rectocolitis). During the routine screening visit, all individuals underwent, among other exams, a complete blood cells count, and urine and faecal parasitology. Copromicroscopy after formol-ether concentration was performed for intestinal parasites and PCR for the detection of Schistosoma spp DNA in stool (modified from [14]). Urine samples were collected between 10 am and 2 pm, concentrated by filtration, and microscopically evaluated for the presence of S. haematobium eggs and for the performance of Schistosoma antigen CCA urinary ICT (nal von Minden GmbH, Moer, Germany). Anti-Schistosoma serology was performed by ELISA (Bordier Affinity Products, Crissier, Switzerland) and/or IgM-IgG immunochromatographic (ICT) test (LDBio Diagnostics, Lyon, France), according to the manufacturer's instructions. On the same day of the visit, i.e. before laboratory results were known, individuals confirming their participation to the study by signing the informed consent form were investigated by abdominal US (all performed by FT) using a SonoSite M-Turbo (Fijifilm, Washinghton, USA) equipped with convex and linear probes. For the intestinal US investigation, the sigma, proximal part of ascending colon next to the ileocecal valve, and terminal ileum were visualized in the area between the iliac vessels and psoas muscle. The area of maximum gut wall thickness was visually identified in each of these segments and measured in each segment (in millimetres; primary outcome). Care was taken to avoid measurement in correspondence to semilunar folds in the colon and during peristaltic contractions in the ileum. Other abdominal features (presence of abdominal effusion, abnormally dilated bowel loops, enlarged mesenteric lymph nodes, mesenteric hypertrophy) recorded in the case report form, images and videos. Liver characteristics, including image pattern according to WHO [12], and spleen characteristics on US were also evaluated by US and recorded. The spleen size was evaluated using both bipolar length in relation to person's height [15] and cross-section area [16], measured on a longitudinal scan passing through the splenic hilum.
Patients with a diagnosis of S. mansoni infection confirmed by microscopy and/or PCR were re-evaluated by intestinal US one month after completion of therapy with praziquantel (40 mg/kg/day in 2 divided doses for 3 days). This time point was decided based on experience with inflammatory lesions of the bladder mucosa in patients with S. haematobium infection, which either disappear or decrease in size within one month post-therapy in most cases [17][18][19]. Inflammatory indexes (Erythrocyte Sedimentation Rate [ESR, normal value <34 mm/H] and/or C Reactive Protein [CRP, normal value <5 mg/l]) were assessed as part of routine tests, while signs and symptoms at the time of hospitalization for treatment were obtained from clinical documentation.

Statistical analysis
Individuals were classified as follows: 1) patients with S. mansoni infection confirmed by presence of S. mansoni eggs in faeces; 2) patients with S. haematobium infection confirmed by presence of S. haematobium eggs in urine; 3) serological schistosomiasis assessed by at least 1 positive serology test but no detection of eggs in faeces and/or urine; 4) schistosomiasis negative individuals, with no laboratory sign of infection. Patients with confirmed S. mansoni infection were analysed as the cases, while the other groups were considered as controls. Presence of other intestinal parasitic infections was considered a confounding factor and its distribution among groups evaluated. Unfortunately it was not possible to stratify results according to infection intensity, which can be a potential effect modifier, as stool examination by Katokatz, a poorly sensitive technique not useful for diagnosis at individual patient's level, is not part of the routine coproparasitological examination of our hospital. The Shapiro-Wilk test was used to assess the normal distribution of the data. Continuous variables were described by mean and standard deviation (SD) and compared by ANOVA in case of independent groups (case-control study) and by paired T-test in case of paired measures (longitudinal study) respectively. Categorical variables were described by count and percentage and compared by Chi-square test. Analyses were performed by MedCalc software (Ostend, Belgium). A pvalue � 0.05 was considered statistically significant.

Participants characteristics
A total of 107 subjects were eligible and they all accepted to take part to the study. The details of included patients are summarized in

Cross-sectional study
As shown in Fig 2, there was no overtly pathological thickness (>4 mm [20]) of the intestinal wall in investigated areas in patients with S. mansoni infection. No significant differences were found with other groups for any of the investigated intestinal segments: sigma (p = 0.811); ascending colon (p = 0.667); and terminal ileum (p = 0.128). No other pathological abdominal image could be detected by US in patients with confirmed S. mansoni infection or in the other groups.

Other characteristics of S. mansoni infected patients and longitudinal study
At diagnosis, no S. mansoni infected patient had US signs of hepatic portal fibrosis, with the exception of two patients showing a pattern C ("ring echoes and pipe stem" [12]), one of whom also had a "starry sky" appearance of a normal sized spleen. Mild to moderate splenomegaly was observed in 6 patients with S. mansoni infection when the cross-sectional area was used (mean spleen area in splenomegalic patients = 67.33 cm 2 , SD 20.28), while also bipolar size in relation to height was above normal limits in 2 of these patients (15.6 cm and 13 cm in male patients 175 cm and 165 cm tall, respectively). During hospitalization, no blood nor mucus in stool was observed in patients infected with S. mansoni; 3 patients had slight pain to the palpation of the abdomen. Three patients referred "abdominal pain" either presently or in the past, and 2 reported "occasional red blood in stool in the past". However, the cultural and linguistic barriers often present limit the reliability of these data.
ESR was within normal limits in all patients (mean 11 mm/H, SD 6); CRP was normal (1.37 mg/l, SD 0.61) in all patients with the exception of one (8.81 mg/l) who also had splenomegaly and abdominal pain.
The maximum intestinal wall thickness in each investigated segment was measured one month after treatment with praziquantel in 11 out of the 17 patients with S. mansoni infection. There was no significant change in maximum wall thickness of the sigma (p = 0.066), ascending colon (p = 0.066), and terminal ileum (p = 0.101) one month after treatment (Fig 3) and calprotectin most likely indicates rapid mucosal integrity restoration after treatment [9]. Although these laboratory tests could be used as markers of S. mansoni intestinal morbidity, non-invasive assessment of intestinal pathology, as it could be provided by US, would allow a more comprehensive assessment of morbidity before and after treatment at both individual patient level in the clinical setting and in community studies. US reports on intestinal schistosomiasis, however, are extremely scant. Dittrich and colleagues [13] performed intestinal US on 173 individuals in an endemic area of Senegal, reporting intestinal abnormalities in 82% of patients with documented (Kato-Katz positive) S. mansoni infection and 60% of uninfected patients, with an apparent positive correlation between infection intensity (eggs per gram of faeces) and prevalence of abnormalities on US. These included intestinal wall thickening, pseudo-kidney or cockade-like images, and increased echogenicity of the mucosa and of the mesenteric structures. None of these abnormalities were detected in our cohort. Many factors (endemic vs non-endemic area, inclusion vs exclusion of children, different intensity of infections in the two cohorts, different coproparasitological diagnostic method used, prevalence and type of concomitant intestinal infections, image resolution of the US machine and of the probe used-convex low-frequency probe by Dittrich and colleagues vs linear high-frequency highly accurate probe in our study) could have contributed to the very different results of the two studies. Also, the reported very high prevalence of abnormal intestinal findings in individuals apparently not infected with S. mansoni supports the hypothesis that the two study populations were markedly different, and therefore difficult to compare. However, a common finding was the lack of visualization of polypoid formations, which conversely are detected in infected patients by colonoscopy [22]. This is consistent with the very low sensitivity of unenhanced US for this type of lesions. Sensitivity could be increased by the use of hydrocolonic sonography [27], however, this technique is time consuming, generally poorly accepted, and not suitable for large-scale studies.
The absence of visualization of intestinal wall thickening in our cohort of patients eliminating S. mansoni eggs in faeces was surprising, although this result should be considered preliminary due to the small sample size. The absence of difference between cases and controls in intestinal wall thickness, with this being overall within normal limits in all groups, counters the possible bias deriving from including S. mansoni infected individuals in other control groups due to low sensitivity of diagnostic methods. It is possible to speculate that only minimal inflammation was expressed in the examined patients. This is supported by the normal systemic inflammation markers in almost all patients, as well as by the absence or low prevalence of clear signs of infection at diagnosis and reported current or past signs and symptoms. These, however, are known to have low sensitivity in infected individuals [28] and collection of an accurate clinical history was often hampered by cultural and linguistic barriers. Unfortunately local markers such as faecal occult blood and calprotectin are not routinely tested in patients diagnosed with schistosomiasis in our centre, and were therefore not available. Another limitation of our study is the unknown intensity of infection in terms of eggs per gram of faeces, as routine copromicroscopical examination in our centre is carried out using formol-ether concentration and not Kato-Katz. Several epidemiological and intervention studies found a correlation between S. mansoni infection intensity and morbidity, including portal fibrosis [29][30][31][32][33][34]. Considering the overall clinical picture of the patients involved in our cohort, it is therefore plausible that our cohort of S. mansoni infected patients may have included only subjects with low intensity infections.
In conclusion, we carried out for the first time a pilot, prospective case-control study of intestinal unenhanced US for S. mansoni morbidity assessment outside endemic areas, using a standard protocol and high-resolution US equipment. Our preliminary results suggest that, although appealing, intestinal US is not likely to be a useful tool in all settings for a more comprehensive assessment of morbidity before and after treatment for S. mansoni infection. Further studies are needed to describe and assess the usefulness of intestinal US on different populations, such as in endemic areas and in patients stratified by infection intensity. Ultrasound findings may be compared with the results of fecal calprotectin and fecal occult blood tests [9]. In the hospital setting, studies comparing colonoscopy and ultrasonography could be envisaged.
Supporting information S1