Schistosoma mansoni antigen detects Schistosoma mekongi infection

Schistosoma mansoni antigen detects Schistosoma mekongi infection

Acta Tropica 141 (2015) 310–314 Contents lists available at ScienceDirect Acta Tropica journal homepage: Schist...

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Acta Tropica 141 (2015) 310–314

Contents lists available at ScienceDirect

Acta Tropica journal homepage:

Schistosoma mansoni antigen detects Schistosoma mekongi infection Beatrice Nickel a,b,∗ , Somphou Sayasone c , Youthanavanh Vonghachack a,b,d , Peter Odermatt a,b , Hanspeter Marti a,b a

Swiss Tropical and Public Health Institute, Basel, Switzerland University of Basel, Basel, Switzerland National Institute of Public Health, Vientiane, Lao Democratic People’s Republic d University of Health Sciences, Faculty of Medicine, Vientiane, Lao Democratic People’s Republic b c

a r t i c l e

i n f o

Article history: Available online 10 August 2014 Keywords: Schistosoma mekongi Schistosoma mansoni Serodiagnosis ELISA Kato-Katz Laos

a b s t r a c t Northern Cambodia and Southern Laos are highly endemic for Schistosoma mekongi. However, there is currently no immunological assay available that is specific for this form of schistosomiasis. We have validated Schistosoma mansoni antigens to detect S. mekongi-directed antibodies in human sera collected from a highly S. mekongi endemic region in Laos. On two consecutive days stool samples of 234 individuals were analyzed by Kato-Katz for presence of S. mekongi eggs and the results were correlated with serology. A sensitivity of 94.5% was calculated for a combination of ELISA and indirect fluorescence assay (IFA) as compared to the detection of S. mekongi eggs in stool samples as gold standard. The results demonstrate that S. mansoni antigens can be used for the diagnosis of S. mekongi infections. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Schistosomiasis is a widely distributed trematode infection in tropical regions with an estimated >200 million people infected. Schistosoma mansoni, Schistosoma haematobium and Schistosoma intercalatum occur in Africa and South America, whereas Schistosoma japonicum and Schistosoma mekongi are the causative agents of intestinal schistosomiasis in Asia. S. mekongi is endemic in two provinces in northern Cambodian (Kratié and Stung Treng) and in the most southern Champasack Province in Lao People’s Democratic Republic (Lao PDR, Laos) (Muth et al., 2010). 150,000 people are estimated at risk of infection (Urbani et al., 2002). Foci of intense transmission are communities of the Mekong island areas of Southern Laos (Sayasone et al., 2011, 2012). In the recent decade, the Mekong river area has emerged as an attractive destination for travellers. Several case reports of Mekong schistosomiasis in travellers have been published in the last decade,

Abbreviations: AWE, adult worm antigen extract from S. mansoni; EPG, eggs per gram Kato-Katz stool examination; SEA, soluble egg raw antigen from S. mansoni; STH, soil-transmitted helminth infection; IFA, indirect fluorescence assay; DDIA, dipstick dye immune assay. ∗ Corresponding author at: Medical Department and Diagnostics, Swiss Tropical and Public Health Institute, CH-4002 Basel, Switzerland. Tel.: +41 61 284 82 44. E-mail address: [email protected] (B. Nickel). 0001-706X/© 2014 Elsevier B.V. All rights reserved.

some of them documenting severe sequelae (Clerinx et al., 2013; Houston et al., 2004; Leshem et al., 2009). In the S. mekongi endemic area diagnosis is mainly based on stool examination using standard parasitological techniques like Kato-Katz (Ohmae et al., 2004). This has a high specificity and only detects active infections, but the sensitivity is low and light infections are frequently missed (Utzinger et al., 2011). Examination of multiple stool samples with at least duplicate Kato-Katz thick smears per sample is recommended (Lovis et al., 2012), but cannot fully compensate for the low detection limit of the method. Immunological assays promise a higher sensitivity by detecting antibodies or antigens. For detection of Schistosoma-specific antibodies ELISAs employing soluble egg antigens (SEA) of S. mansoni (van Gool et al., 2002) or S. japonicum (Zhu, 2005), as well as cercarial antigen preparations (cercarial transformation fluid, SmCFT) are being used (Smith et al., 2012). To date, no specific serological diagnosis is available for S. mekongi. Development of a specific assay would imply production of S. mekongi antigen, which is hampered by the challenging rearing of Neotricula aperta, the intermediate snail host of the parasite. The use of cross-reacting antigens from other Schistosoma species would be an alternative. ELISAs using S. japonicum SEA for detection of S. mekongi antibodies were successfully used in control programmes in the Mekong River basin (Ohmae et al., 2004) and the dipstick dye immune assay (DDIA) with S. japonicum SEA showed a sensitivity of more than 97% for detection of S. mekongi infections (Zhu et al., 2005). A serodiagnostic assay using adult worm antigen

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extract from S. mansoni; (AWE) and SEA of S. mansoni for detection of Schistosoma-specific antibodies is routinely performed at the diagnostic centre of the Swiss Tropical and Public Health Institute (Swiss TPH) with a sensitivity for S. mansoni of 98% and 80% for AWE and SEA, respectively, and a specificity of 96% and 92% (unpublished data). Positive or equivocal results in one or both tests are always further examined with a confirmatory Schistosoma indirect fluorescence assay (IFA). Both antigens show cross-reactivity with antibodies elicited by S. haematobium or S. japonicum infections. In our study we tested the S. mansoni antigen preparations for the detection of S. mekongi-specific antibodies. We validated the S. mansoni antigen-based serology for the diagnosis of S. mekongi infections in a cross-sectional study in Southern Laos using the results of Kato-Katz stool examinations as gold standard.


carried out for each stool sample by experienced technicians. Thirty minutes clearance time was given between smear preparation and reading of the slides. Eggs were counted and noted for each parasite species separately. 2.4. Serum collection and examination From each study participant a 5 ml blood sample was drawn. At the health centre of Don Khon the coagulated blood was centrifuged and separated serum was frozen in a −20 ◦ C freezer. Aliquots of the serum samples were sent frozen to the Diagnostic Centre of the Swiss TPH in Basel, Switzerland, where they were stored at −80 ◦ C until assayed. Samples were defrosted in maximum twice for performing the assay. 2.5. Preparation of antigen

2. Method and population 2.1. Ethics statement This study was integrated in a larger study on multi-parasitic infections and their control approved by the Lao National Ethics Committee for Health Research (NECHR), Ministry of Health, Lao PDR. Study participants were informed about the study procedures, the benefits and risks as well as their voluntary participation. Before enrolment written informed consent was obtained from each study participant and/or parents or legal guardians of children below the age of 15 years. In addition a written assent was obtained from children and adolescent below age of 18 years. Participants were informed about the examinations. All infections diagnosed were treated according to the Lao National treatment guidelines (MOH, 2004), i.e. S. mekongi infection was treated with praziquantel (40 mg/kg BW, single oral dose). 2.2. Study design and area Between October 2011 and August 2012 a cross-sectional study was conducted in four villages of two Mekong islands, Don Khon (Ban Khon and Ban Hang Khon village) and Don Som (Som Ven Ook and Ban Yai Veun Som village) in Southern Khong district, Champasack Province, Laos. Twenty-five households were randomly selected from each village. All household members older than 2 years were eligible to participate in the study. For the current study, samples of 234 individuals of the crosssectional study were selected for detection of S. mekongi antibodies using S. mansoni antigens. The Khong district is highly endemic for helminth infections. Recent studies have shown that in certain villages S. mekongi infection may reach 50% or more (Sayasone et al., 2011, 2012). Opisthorchis viverrini and minute intestinal flukes (MIF) such as Haplorchis taichui are most frequent (Chai et al., 2013). Soiltransmitted helminth (STH) infection such as hookworm, Ascaris lumbricoides and Trichuris trichiura may reach high infection rates (Eom et al., 2014). Recently it was demonstrated that Strongyloides stercoralis infection prevalence may reach 40% and higher (Vonghachack et al., 2014). Taenia spp. (Jeon et al., 2013) and Gnathostoma spp. (Vonghachack et al., 2010) infections were also reported. The district is endemic for Plasmodium falciparum malaria. 2.3. Field procedures and stool examinations From each study participant two stool samples were collected and examined using Kato-Katz thick smears technique (Katz et al., 1972). Pre-labelled plastic stool containers were handed out to each participant. The following morning, filled containers were collected and replaced by empty ones for collection on the following day. In the Health Centre of Done Khon, two Kato-Katz thick smears were

Adult worm antigen extract (AWE): Adult S. mansoni worms were homogenized in PBS (pH 7.2), 2 mM PMSF for 1 h at 4 ◦ C and the extract was centrifuged at 80,000 × g for 3 h at 4 ◦ C. The pellet was re-suspended and homogenized in PBS (pH7.2) containing 1% NP-40, 2.5 mM EDTA, 0.2 mM TPCK, 0.2 mM TLCK, 1 mM o-phenantroline, 2 mM PMSF, 0.05 mg/ml SAM chloride dihydrochloride, 0.05 mg/ml leupeptin and 0.05 mg/ml chymostatin. After overnight incubation on a stirrer at 4 ◦ C the suspension was centrifuged at 80,000 × g for 3 h at 4 ◦ C and the resulting supernatant was further concentrated in an Amicon stirred cell (model 402, Millipore corporation) using an Ultracel disc membrane (YM30). Concentrated antigen was centrifuged at 15,300 × g for 5 min at 4 ◦ C, and the supernatant was stored in aliquots at −80 ◦ C until use. Soluble egg antigen (SEA): Frozen S. mansoni eggs were homogenized in PBS (pH 7.2) on ice, subsequently extracted for 3 h on a stirrer at 4 ◦ C and the extract then centrifuged at 100,000 × g for 2 h at 4 ◦ C. Supernatant was stored in aliquots at −80 ◦ C until use. 2.6. Screening ELISA For serodiagnosis two in-house ELISAs were carried out, one using S. mansoni AWE and the other one using SEA as described (Junghanss and Weiss, 1992). In brief, antigens were coated in 0.05 M sodium carbonate buffer, pH 9.6, to Immulon 2HB plates (Thermo Labsystems, 735-0462). After washing, diluted sera were added to the plates and incubated for 15 min at 37 ◦ C. After additional washing steps, horseradish peroxidase conjugated goat-anti-human-IgG (KPL, 474-1006) was added. Plates were incubated for 15 min at 37 ◦ C, subsequently washed and o-Phenylendiamine Dihydrochloride (OPD, Sigma) was added. Reaction was stopped with 8 M H2 SO4 and absorption was read with a Multiscan FC reader (Thermo Scientific) at 492 nm. All sera giving positive or equivocal results were additionally tested with an in-house confirmatory Schistosoma IFA. 2.7. Schistosoma IFA Adult male Schistosoma worms were washed in 0.9% NaCl, packed closely and shock-frozen in petroleum ether with dry ice. Thin sections were cut with a cryomicrotome and mounted on glass microscope slides. Slides were stored at −80 ◦ C until further use. For IFA analysis slides were quickly air-dried and fixed with acetone. Diluted sera were then applied to the slide-slots. Three control sera were present on each slide, one positive, one equivocal and one negative control. After 25 min of incubation at 37 ◦ C, slides were washed with PBS and air-dried. FITC conjugated F(ab)’2 anti-IgG/A/M (BioRad, #30244) diluted in 0.01% Evansblue in PBS was added, slides were incubated for 25 min at 37 ◦ C, washed, dried and a cover glass was mounted with buffered glycerol. Slides were


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Table 1 Cutoffs for the S. mansoni ELISA and IFA, and scheme for final interpretation. Antigen

ELISA cutoff (OD)





<0.15 <0.3

0.15–0.29 0.30–0.59

≥0.3 ≥0.6

IFA cutoff (reciprocal titre)








Overall interpretation of serology








AWE = adult worm extract; SEA = soluble egg antigen.

examined immediately with a fluorescence microscope. The IFA was considered positive when reactive at a serum dilution ≥1:160, equivocal at a dilution of 1:80 and negative when lower.

Fig. 2. Schistosoma mekongi infection intensity (EPG) grouped by serological results (n = 234). *The group with positive serology comprises 3 samples >300 EPG (not shown in the figure).

3. Results 2.8. Data analysis All data was double entered in EpiData version 3.1 (EpiData Association, Odense, Denmark) and validated. Analyses was performed in STATA version 10 (StataCorp., College Station, USA) and Excel. The cumulative results of the stool examination (two stool samples each with two Kato-Katz smears, total 4 smears) were considered as gold standard. The results of the ELISAs in this study were interpreted according to the cutoffs previously determined with sera from healthy Swiss blood donors and sera from S. mansoni infected patients (Table 1). For serum samples which showed positive OD492 values for both, AWE and SEA, as well as sera which were negative in both ELISAs, no additional IFA was performed. All other samples were subsequently tested with an IFA. The final interpretation based on the results of all three tests was interpreted according to the following criteria: negative if all tests were negative or if two tests were negative and one test was equivocal; positive if at least two out of three tests were positive; all other combinations were interpreted as equivocal. Sensitivity and predictive values were calculated using the results of the stool examination as gold standard. The true prevalence of S. mekongi infections within the 234 individuals was calculated from the results of the stool analysis using a mathematical model (Marti and Koella, 1993).

Fig. 1. Serological results in Schistosoma mekongi stool positive and stool negative participants.

3.1. Study population Stool and serum samples were collected from 234 participants; 124 females (53%) and 110 males (47%). The mean age was 25 years (range 2–77 years) and was not significantly different among the sexes. When the stool samples were examined microscopically by multiple Kato-Katz 183 individuals (78.2%) were diagnosed with S. mekongi infection (Fig. 1). There was no significant difference between gender (male 81.6% vs female 75.2%, p = 0.268). The mean infection intensity was 48 EPG (range 6–1110 EPG). Three patients had an infection intensity above 300 EPG (Fig. 2), namely 382 EPG, 582 EPG and 1110 EPG. O. viverrini (68.8%) and hookworm (49.6%) were diagnosed in considerable frequencies while T. trichiura (4.7%) and A. lumbricoides (0.4%) were detected only in a few individuals. 3.2. Validity of S. mansoni ELISA for the detection of S. mekongi The 234 samples were tested serologically with ELISA on two different antigen preparations, AWE and SEA. Of the 183 Kato-Katz positive individuals, 123 sera tested positive with both antigens, while 8 sera were negative with both antigens. These samples were interpreted as positive or negative, respectively and not further tested. The other 52 samples, which had shown at least one equivocal result, were additionally tested in the IFA. The final interpretation of the test result was carried out according to Table 1. A total of 173 Kato-Katz positive samples were positive in serology and 10 samples were negative. The calculated sensitivity of the S. mansoni-based serology for detection of S. mekongi antibodies was 94.5%. Out of 51 Kato-Katz negative samples, 31 were positive in serology and 20 were negative (Fig. 1). As no sufficient number of non-infected individuals from the same region who had never been exposed to schistosomiasis was available, the specificity could not be calculated. Taking the Kato-Katz results as gold standard, the calculated PPV and NPV were 84.8% and 66.7%, respectively. The calculated prevalence of S. mekongi infection within the tested population was 87%, as compared to the measured prevalence of 64% (Marti and Koella, 1993). Although serology does not exclusively detect on-going infections, a significant correlation of serological reactivity and infection intensity, as reflected by the number of eggs per gram of stool (EPG), could be demonstrated (Fig. 2). For the 143 samples with a positive

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serology the mean EPG was 52. For the 61 samples with an equivocal result the mean EPG was 22, and the stool samples of the 30 serology-negative individuals showed a mean EPG of 4 including 20 stool samples without any eggs (Kruskal–Wallis test, p = 0.001).

4. Discussion The detection of Schistosoma-infected individuals in endemic areas relies on microscopic demonstration of eggs in the stool, usually after concentration according to the Kato-Katz method (MOH, 2004; WHO, 1995). Although this method is cheap and easy to perform, it lacks sensitivity (Knopp et al., 2009). The detection of specific antibodies, and lately also of antigens in travellers returning from endemic countries, are valuable alternatives in specialized laboratories (Cavalcanti et al., 2013). Currently the antigens used for serology are mostly raw extracts of adult worms (AWE) or eggs (SEA) of S. mansoni or S. japonicum. Other Schistosoma species or their snail intermediate hosts, like Neotricula spp., are difficult to keep in the laboratory for prolonged periods of time and therefore maintaining the life cycle is challenging. S. mansoni antigens show cross-reactivity with S. haematobium and S. japonicum specific antibodies (Smith et al., 2012), although the sensitivity is lower than for S. mansoni sera. Here we tested the cross-reactivity of S. mekongi sera with S. mansoni antigens and for the first time validated the sensitivity of this test system for S. mekongi. We demonstrate that S. mansoni antigens detect S. mekongi infections with a high sensitivity. Out of 183 patients with S. mekongi infection as revealed by microscopy, 173 showed a positive serology, resulting in a sensitivity of 94.5%. Although a positive serology is not necessarily linked to an active infection, we observed that higher infection intensities also showed significantly higher titres in the serological examination (Fig. 2). Out of the 10 serologynegative results, 8 sera showed no reaction in both ELISAs, while 2 reacted equivocally with one antigen, but could not be confirmed by the IFA. Regulatory mechanisms of the parasite on the immune system of the host are known in patients with chronic S. mansoni infections, which might explain the negative serology (Colley et al., 1986). Negative regulatory effects are not observed in the acute phase of infection (Colley et al., 1986). Cases of negative serology in egg-positive individuals were also described for S. mansoni and S. haematobium infections, where stool or urine positive individuals did not seroconvert within 6 months. In some other cases seroconversion occurred only after 4–6 months, while eggs were detected much earlier (Bottieau et al., 2006; Soentjens et al., 2014). The authors describe that Schistosoma hybrid eggs were identified by microscopy and PCR revealed S. haematobium–S. bovis hybrids in these individuals (Soentjens et al., 2014). Existence of such hybrid eggs might be the reason for suboptimal serology results. Further evaluation of such cases in Laos with molecular techniques would be important to monitor if hybrids also occur in S. mekongi endemic area. In the current study only 51 Kato-Katz negative samples could be assessed, all from individuals originating from the same community and having the same exposure risks as the positive individuals. No sampling of healthy individuals from the same area, who had never experienced a Schistosoma infection, could be carried out and the analysis of possible cross-reactivity is therefore difficult. In 31 individuals of 51 negative by Kato-Katz, Schistosomaspecific antibodies were demonstrated. As these individuals living in the endemic area had the same high probability of acquiring an infection as Kato-Katz positive individuals, it is possible that the infection was either not detected by the microscopic examination with the relative insensitive Kato method (Knopp et al., 2009), or the persons exhibited a persistent antibody titre due to a former infection which had been treated before the study at


the local health facility. Antibody titres may persist for months or even years after successful treatment (Vendrame et al., 2001) and a serological test carried out after successful treatment within that time-frame would still yield a positive result. In view of these facts and the absence of a truly “healthy” control group, the calculation of the specificity of the test could not be carried out in this study population. The next step will be to apply the serological test with S. mansoni antigens to travellers returning to Europe or the US from S. mekongiendemic areas. This will allow to validate the test for detecting recently acquired infections. Knowledge of the exposure history of these patients will then also allow to calculate the specificity of the test. 5. Conclusion Further investigations into the specificity of the test for detection of anti-S. mekongi antibodies are necessary by testing healthy individuals from the same region never infected with S. mekongi. Clarification of cross-reactivity to other helminthic infections will be required to give insight into the specificity of the assay in this region, especially in view of the relatively high infection rates with O. viverrini and hookworms. In addition, molecular investigations of eggs found in serological negative individuals would disclose if Schistosoma hybrids occur, as observed by Soentjens et al. (2014) for S. haematobium–S. bovis hybrids in Mali. Our results suggest that the use of S. mansoni antigens for serological testing for S. mekongi infection is a valid diagnostic approach with high sensitivity. The potential of the test for detection of S. mekongi infections in travellers returning from endemic areas remains to be established. Acknowledgements The field data collection was supported by International Development Research Centre; Foreign Affairs, Trade and Development Canada (through the Global Health Research Initiative); and the Australian Agency for International Development (The Project No. 105509-027). We thank Christina Krebs from the Diagnostic Centre, Swiss TPH, for expert technical assistance and Pascal Mäser from the Department of Medical Parasitology and Infection Biology, Swiss TPH, for critical reading. References Bottieau, E., Clerinx, J., de Vega, M.R., Van den Enden, E., Colebunders, R., Van, E.M., Vervoort, T., Van, G.A., Van den Ende, J., 2006. Imported Katayama fever: clinical and biological features at presentation and during treatment. J. Infect. 52, 339–345. Cavalcanti, M.G., Silva, L.F., Peralta, R.H., Barreto, M.G., Peralta, J.M., 2013. Schistosomiasis in areas of low endemicity: a new era in diagnosis. Trends Parasitol. 29, 75–82. Chai, J.Y., Yong, T.S., Eom, K.S., Min, D.Y., Jeon, H.K., Kim, T.Y., Jung, B.K., Sisabath, L., Insisiengmay, B., Phommasack, B., Rim, H.J., 2013. Hyperendemicity of Haplorchis taichui infection among riparian people in Saravane and Champasak Province, Lao PDR. Korean J. Parasitol. 51, 305–311. Clerinx, J., Cnops, L., Huyse, T., Tannich, E., Van, E.M., 2013. Diagnostic issues of acute schistosomiasis with Schistosoma mekongi in a traveler: a case report. J. Travel. Med. 20, 322–325. Colley, D.G., Garcia, A.A., Lambertucci, J.R., Parra, J.C., Katz, N., Rocha, R.S., Gazzinelli, G., 1986. Immune responses during human schistosomiasis. XII. Differential responsiveness in patients with hepatosplenic disease. Am. J. Trop. Med. Hyg. 35, 793–802. Eom, K.S., Yong, T.S., Sohn, W.M., Chai, J.Y., Min, D.Y., Rim, H.J., Jeon, H.K., Banouvong, V., Insisiengmay, B., Phommasack, B., 2014. Prevalence of helminthic infections among inhabitants of Lao PDR. Korean J. Parasitol. 52, 51–56. Houston, S., Kowalewska-Grochowska, K., Naik, S., McKean, J., Johnson, E.S., Warren, K., 2004. First report of Schistosoma mekongi infection with brain involvement. Clin. Infect. Dis. 38, e1–e6. Jeon, H.K., Yong, T.S., Sohn, W.M., Chai, J.Y., Min, D.Y., Yun, C.H., Rim, H.J., Pongvongsa, T., Banouvong, V., Insisiengmay, B., Phommasack, B., Eom, K.S., 2013. Current status of human taeniasis in Lao People’s Democratic Republic. Korean J. Parasitol. 51, 259–263.


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