Am. J. Trop. Med. Hyg., 79(3), 2008, pp. 422-426
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene
Eosinophil Cationic Protein, Soluble Egg Antigen, Circulating Anodic Antigen, and Egg Excretion in Male Urogenital Schistosomiasis
Peter D. C. Leutscher*,
Govert T. J. van Dam,
Claus M. Reimert,
Charles-Emile Ramarakoto,
André M. Deelder, AND
Niels Ørnbjerg
DBL Centre for Health Research and Development, Institute of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark; Department of Parasitology, Leiden University Medical Centre, Leiden, The Netherlands; Department of Epidemiology, Institute Pasteur, Antananarivo, Madagascar
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ABSTRACT
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Markers of male genital schistosomiasis (MGS) are needed to elucidate the consequences for reproductive health. Eosinophil cationic protein (ECP) and soluble egg antigen (SEA) in urine and semen, and circulating anodic antigen (CAA) in serum were assessed as MGS markers. Egg counts, ECP, and SEA in urine and CAA in serum, correlated positively. Seminal egg excretion exhibited marked day-to-day variations, but counts correlated positively with urinary egg counts and SEA in semen and with CAA. Positive predictive values with reference to seminal egg excretion were as follows: seminal ECP (52%), seminal SEA (83%), CAA (97%), and urinary egg excretion (82%). SEA in semen and CAA in serum constitute potential markers of MGS. However, urine egg counts as an indirect marker of MGS remains the preferred diagnostic method from a public health perspective.
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INTRODUCTION
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Egg-induced inflammation and development of fibrosis may occur at any anatomic site of the pelvic region in Schistosoma haematobium–infected persons. A rich system of venous anastomosis enables the adult worm pair, especially after puberty, to migrate to the genital organs from the venous plexus of the urinary bladder.1 Post-mortem studies have thus confirmed that eggs are as frequently found in the genital organs as in the bladder in men and women.2–5 The prostate gland and the seminal vesicles are particularly affected in men. Male genital schistosomiasis (MGS) has in this context been hypothesized to be a be risk factor for transmission of human immunodeficiency virus (HIV) because of egg-induced inflammation in the seminal fluid–producing organs and an associated potentially increased viral shedding in ejaculate from co-infected persons.6 Female genital schistosomiasis (FGS) is likewise considered to be a risk factor for HIV transmission because of egg-induced lesions in the lower genital tract, making the S. haematobium-infected women more susceptible to passage of virus over the damaged mucosa barrier.7
In general, epidemiologic and clinical data regarding genital schistosomiasis and associated reproductive health morbidity are sparse. This is, in part, because of a lack of validated markers for genital infection. Urine egg counts, the traditional marker of urinary S. haematobium infection, may not necessarily be applicable as a marker for infection of the genitals. A cross-sectional study of FGS in Tanzania showed that 25% of 263 women detected with eggs in cervical tissue samples were egg negative in repeated urine samples.8 Similarly, in an autopsy study, a significant proportion of men with S. haematobium infection had egg-positive genital tissue but were negative for eggs in bladder tissue.2
Eosinophil cationic protein (ECP) is an established marker of urinary S. haematobium infection and morbidity.9–11 This protein has also been evaluated as an infection marker for genital schistosomiasis.12–14 Preliminary results showed a positive association between egg excretion and increased ECP levels in ejaculates.14 A positive association between S. haematobium eggs in cervical biopsy specimens and smears and increased ECP levels in vaginal lavage has also been observed.13
The aim of this study was to further investigate ECP and soluble egg antigen (SEA) in urine and semen, in addition to circulating anodic antigen (CAA) in serum, an adult schistosome excretion product, as markers of MGS. CAA and SEA have both proven highly useful as pre- and post-therapeutic infection markers for urinary schistosomiasis.15–19
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MATERALS AND METHODS
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Population
This study was conducted in the Sirama sugarcane plantation 25 km from Ambilobe in northern Madagascar. This area is endemic for S. haematobium and has shown an overall prevalence of more than 50% (Leutscher PDC, unpublished data). Study participants were recruited from a catchment area in the plantation with 2,663 residents. All male residents 15–49 years of age who were egg positive for S. haematobium in urine, as demonstrated by filtration of one urine sample, were invited to participate in the study. Adult males of the same age group from an area with low endemicity, Mataipako, 32 km south of Sirama, with a population of 865 residents, served as controls if egg negative in urine. An unpublished survey conducted five years before the present study showed an overall S. haematobium prevalence of less than 20% in the village. Socioeconomic factors and access to health care were comparable in the two study areas. No systematic anti-schistosome therapy had been provided previously in the study areas, but a history of treatment was reported by a few study participants. After sampling, participants and the general population in the two study areas were mass-treated with praziquantel (40 mg/kg). The study was reviewed and approved by the Ministry of Health and the National Committee of Ethics in Madagascar.
Parasitology
Egg counts per 10 mL of urine were determined by filtration of two urine samples obtained on different days using a Nucleopore® membrane (Costar Co., Cambridge, MA). Egg count was estimated as mean egg count of two samples. Two semen samples from each study participant taken two weeks apart were requested. However, participants who provided only one sample were also included. Although the study participants were asked to refrain from sexual activity for three days before collection of semen samples, there was no verification of the compliance with that request. When two samples were provided, the highest of the two egg counts was used. Participants could ejaculate either into a dry plastic container or a non-spermicidal condom (Seminal Collection Device®; HDC Corporation, Milpitas, CA). Subsequently, the egg count per total ejaculate volume was measured by filtration of semen when liquefaction was completed by using a Whatman® membrane (Whatman International Ltd., Maidstone, United Kingdom) as described.12 For assessment of S. mansoni infection, a Kato thick smear was prepared from one fresh stool sample from each participant obtained at baseline.
Eosinophil cationic protein
Urinary and seminal concentrations of ECP were measured by means of an ECP enzyme-linked immunosorbent assay (ELISA).19 Briefly, the method is a polyclonal sandwich-type ELISA with a biotin-avidinperoxidase amplification step. It measures ECP in the range of 15–1,000 pg/mL. Eosinophil cationic protein purified from extracts of human blood eosinophils was used as a standard. Before measurement, 100 µL of the sample was mixed with an equal volume of 1% N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB) in 0.15 M NaCl, mixed vigorously, incubated for one hour at room temperature, mixed with 0.8 mL of sample buffer (0.1% Tween 20, 0.1% CTAB, 20 mM EDTA, 0.2% human serum albumin, and 0.1% NaN3 in phosphate-buffered saline, pH 7.4), and frozen at –20°C until used for further analysis. Before analysis, extracts were centrifuged (3,000 x g for 10 minutes) and the supernatants containing the extracted proteins were removed and used for ECP analysis. Samples testing beyond a standard curve were further diluted in sample buffer and retested. A cut-off value for ECP of 5 ng/mL of urine has been found to give the test an optimal overall diagnostic efficiency of 90% with a sensitivity of 95% and a specificity of 0.71% (Reimert CM, unpublished data). Because a cut-off value for seminal ECP has not yet been established, the same cut-off value as for ECP in urine was used.
Soluble egg antigen
A monoclonal antibody (MAb)–based sandwich ELISA was used to quantify SEA in urine and semen as described by Kahama and others.20 Briefly, MAb-coated plates were incubated with heat-treated urine or semen samples and SEA standards. After thorough washings, plates were incubated with biotin-labeled MAb, streptavidin–alkaline phosphatase, and p-nitrophenyl phosphate substrate. Absorbance was measured overnight at 405 nm. The lower detection limit for the ELISA was 10 ng/mL (buffers values plus 2 SD). The cut-off value for urine samples was 30 ng/mL, which was obtained from 10 urine samples from persons from an area not endemic for schistosomiasis. The cut-off value for semen samples was 46 ng/mL, which was obtained from 24 negative Dutch donors. Using these cut-offs, we determined that the specificity was 95%.
Circulating anodic antigen
Serum CAA concentrations (pg/mL) were determined by an MAb-based antigen-capture ELISA described by Polman and others with minor modifications.21 A total of 40 pg of CAA/mL was used as a cut-off value, which gave a specificity of 98–100% on the basis of 300 serum samples from different area not endemic for schistosomiasis. Urinary and seminal egg counts were used as reference values in estimation of sensitivity and specificity for serum CAA in this study population.
Statistical analysis
Geometric mean levels for ECP, SEA, and CAA were estimated from samples with detectable levels above the specific cut-off values. The chi-square test was used for comparison of relative frequencies. At baseline, egg counts and CAA, SEA, and ECP levels were compared by the Mann-Whitney ranking test for unpaired data. The same variables were also assessed by Spearman’s rank correlation coefficient (
) test. Wilcoxon’s signed rank test was used for analysis of follow-up data. Geometric means were determined from only positive samples. For determination of sensitivity, specificity, and positive predictive values of ECP and SEA in urine and CAA in serum, detection rates from egg-positive urine samples in Sirama and from egg-negative urine samples in Mataipako were used. The same procedure was used for ECP and SEA in semen and CAA in serum in relation to egg findings in ejaculates.
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RESULTS
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Parasitology
Urine
In Sirama, 173 (62%) of 278 men examined with a median age of 27 years (range, 15–49 years) were positive for S. haematobium eggs in urine. The geometric mean egg count was 13 eggs/10 mL of urine (95% confidence interval [CI], 1–352 eggs/10 mL of urine) (Table 1
). One hundred thirty-one (76%) men had mean egg counts of 1–49 eggs/10 mL of urine, and 42 (24%) men had counts 
50 eggs/10 mL of urine. In Mataipako, urine samples from 128 of 152 eligible men were examined. Thirty-nine (31%) men were egg positive in urine with a geometric mean egg count of 4 eggs/10 mL of urine (95% CI, 1–49 eggs/10 mL of urine). The remaining 89 men with a median age of 28 years (range, 17–47 years) who were negative for eggs in urine were enrolled in the study.
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TABLE 1 Comparison of baseline results between men in Sirama, with positive urine egg excretion and men in Mataipako with negative egg excretion
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Semen
In Sirama, semen samples were provided by 117 men, of whom 49 (42%) were egg positive (Table 1
). The geometric mean count in egg-positive semen samples was 6 eggs/ejaculate (95% CI, 1–51 eggs/ejaculate). Among 67 men who provided two semen samples, 32 were egg positive with the following distribution: 20 (63%) men were positive in only one of two samples and 12 (37%) men were positive in both samples. Sixty-three (71%) men in Mataipako provided semen samples. Seven (11%) men were egg positive with a mean egg count of 2 eggs/ejaculate (95% CI, 1–2 eggs/ ejaculate).
Stool
None of the study participants at the two sites had detectable S. mansoni eggs in the stool.
Eosinophil cationic protein
Urine
In Sirama, 143 (83%) men had detectable ECP concentrations ( 5 ng/mL) in urine with a geometric mean of 82 ng/mL (95% CI, 6–4,573 ng/mL). These values were significantly higher than in Mataipako (Table 1). The ECP concentration and egg count showed a positive correlation ( = 0.304, n = 143, P < 0.001). The ECP positivity rate and geometric mean concentration of ECP were significantly higher in men with egg counts 50 eggs/10 mL of urine than in men with egg counts of 1–49 eggs/10 mL of urine (98% versus 78%; P < 0.01) and in men with ECP levels of 131 ng/mL of urine (95% CI, 6–4,820 ng/mL) than in men with ECP levels of 67 ng/mL of urine (95% CI, 6–1,830 ng/mL; P < 0.05) (Table 2).
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TABLE 2 Levels of eosinophil cationic protein (ECP) and soluble egg antigen (SEA) in urine and circulating anodic antigen (CAA) in serum, according to urine egg count among 173 men in Sirama, Madagascar
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Semen
In Sirama, detectable ECP concentrations ( 5 ng/ mL) in semen was observed in 106 (91%) of the 117 men who provided semen samples (Table 1). The geometric mean in positive samples was 110 ng/mL (95% CI, 6–3,985 ng/mL), which was significantly higher than in egg-negative persons in the Mataipako (Table 1). The ECP positivity rates were not significantly different in egg-positive and egg-negative semen samples in Sirama (Table 3). The geometric means were also comparable in positive samples (ECP level = 176 ng/mL, 95% CI, 10–3,986 ng/mL) and in negative samples (ECP level, 78 ng/mL, 95% CI, 5–619 ng/mL).
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TABLE 3 Levels of eosinophil cationic protein (ECP) and soluble egg antigen (SEA) in semen and circulating anodic antigen (CAA) in serum, according to seminal egg status among 117 semen-donating men in Sirama, Madagascar
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Soluble egg antigen
Urine
In Sirama, 89 (52%) men had detectable SEA concentrations ( 30 ng/mL) with a geometric mean of 331 ng/mL (95% CI, 37–9,061 ng/mL), which was significantly higher than in Mataipako (Table 1). The SEA levels showed a positive correlation with egg counts ( = 0.375, n = 89, P < 0.0001) and ECP levels ( = 0.348, n = 87, P < 0.001). The SEA positivity rate and geometric mean were significantly higher in men with egg counts 50 eggs/10 mL of urine than in men with egg counts of 1–49 eggs/10 mL of urine (83% versus 41%, P < 0.0001) and SEA levels of 747 ng/mL of urine (95% CI, 59–18,266 ng/mL) and in men with SEA levels of 196 ng/mL of urine (95% CI, 37–1,770 ng/mL; P < 0.05) (Table 2).
Semen
Detectable SEA concentrations ( 46 ng/mL) in semen were observed in 29 (25%) men in Sirama with a geometric mean of 540 ng/mL (95% CI, 49–7,120 ng/mL) (Table 1). Values were significantly higher than in Metaipako. In Sirama, the SEA level showed a positive coirrelation with egg count ( = 0.434, n = 29, P < 0.05), but not with ECP level. The positivity rate and the geometric mean were significantly higher in egg-positive semen samples than in egg-negative samples (41% versus 15%; P < 0.02 and 1,129 ng/mL, 95% CI, 84–6,980 ng/mL versus 295 ng/mL, 95% CI, 49–2,512 ng/mL, P < 0.02) (Table 3).
Circulating anodic antiogen
In Sirama, 119 (69%) men had detectable CAA concentrations in serum with a geometric mean of 695 ng/mL (95% CI, 13–11,723 ng/mL) (Table 1). Only one participant in Mataipako was CAA positive. The geometric mean CAA level showed a positive correlation with urinary egg count ( = 0.692, n = 119, P < 10–6) and urinary SEA level ( = 0.398, n = 83, P < 0.0001), but not with urinary ECP level ( = 0.104, n = 112, P not significant). The CAA positivity rate and geometric mean were significantly higher in men with egg counts 50 eggs/10 mL of urine than in men with egg counts of 1–49 eggs/10 mL of urine (93% versus 61%; P < 0.001 and 1,923 ng/mL of urine, 95% CI, 52–27,064 versus 422 ng/mL of urine, 95% CI, 13–4139 ng/mL, P < 0.0001) (Table 2). In Sirama, geometric mean CAA level also showed a positive correlation with seminal egg count ( = 0.345, n = 34, P < 0.05). Thirty-seven (76%) of 49 seminal egg-positive men in Sirama were CAA positive compared with 33 (54%) of 68 egg-negative men (P < 0.02) (Table 3). The geometric mean CAA level was higher in the seminal egg-positive group than in the egg-negative group, but the difference did not reach statistical significance.
Sensitivity, specificity, and positive predictive value
The positivity rates of ECP and SEA in urine and CAA in serum in 173 men from Sirama who were egg positive in urine were compared with those in 89 men from Mataipako who were not excreting eggs. For semen, the comparison was made between 49 men from Sirama who were positive for egg excretion and 56 men from Mataipako with who were negative for egg excretion. The highest sensitivity rates were found for ECP in urine and semen (83% and 97%, respectively), whereas the specificity rates were low (46% and 25%) (Table 4). Both SEA and CAA showed high specificity rates for urine and semen (> 93%), whereas the sensitivity rates were lower (41–76%). Circulating anodic antigen was the strongest predictor of seminal egg excretion, with a positive predictive value of 97%. Urinary egg detection showed a sensitivity of 42%, a specificity of 83%, and a positive predictive value of 82% for eggs in semen.
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TABLE 4 Specificity, sensitivity, and positive predictive value of eosinophil cationic protein (ECP) and soluble egg antigen (SEA) in urine and in semen and circulating anodic antigens (CAA) in relation to Schistosoma haematobium egg-positive urine and semen samples from men in Sirama, Madagascar, and negative samples from men in Mataipako
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DISCUSSION
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A major limitation of the study was that only persons in Sirama who were positive for S. haematobium eggs in urine were enrolled in the study. Additional information would have been obtained if egg-negative persons from this village had been included. Our study simultaneously assessed ECP, SEA, and CAA as markers of infection with S. haematobium. In agreement with results of previous studies,8,10,14,15,17,18 each of the three markers showed a positive correlation with urinary egg counts. Except for ECP and CAA, these markers also showed a positive correlation with each other. The CAA and ECP markers reflect the worm population and egg-induced eosinophil inflammation, respectively, but these factors may not be correlated with each other. However, the correlation between SEA and both ECP and CAA seems plausible because the egg load may be more directly linked to worm burden and intensity of egg-induced inflammation.
The study showed, as previously reported,12 that eggs are frequently found in semen in S. haematobium-infected men. However, seminal egg excretion showed marked day-to-day variation. Thus, 62% of egg-positive men examined twice were egg-negative in one of the two samples. This observation demonstrates that examination of one ejaculate for S. haematobium eggs is inadequate for a proper diagnosis of MGS. However, collection of additional samples would result in further inconvenience for the patients and would not be a feasible, standard, operational procedure for diagnostic purposes.
Post-mortem studies on S. haematobium-infected men have shown that seminal vesicles and the prostate gland are frequent sites of egg deposition as often as the bladder, albeit with lower numbers of eggs per gram of tissue.2–4 However, it was also shown that eggs are more frequently found in the muscle layer than in the mucosa of seminal vesicles. This deeper location may contribute to day-to-day variation in seminal egg excretion because eggs may not easily enter the lumen of the prostate gland and seminal vesicles. Another contributory factor might be that all men did not observe the request for sexual abstinence for three days before semen sampling. Eggs in the lumen of seminal vesicles and prostate gland could theoretically have been ejaculated a short time before the study sample was obtained, resulting in sample would be egg negative.
The variation in seminal egg excretion in our study indicates potential limitations in the use of egg counts as a reference to validate seminal ECP and SEA as markers of MGS infection. In spite of these limitations, seminal SEA, as opposed to seminal ECP, showed a positive correlation with egg counts. The lack of a positive correlation between ECP values and egg counts is consistent with the finding in earlier postmortem studies2 of a lack of a correlation between the number of eggs in male genital tissue and the extent of local inflammation. Few eggs may in some situations cause marked inflammation but many eggs in other situations may cause only mild inflammation. However, a significant higher ECP level was observed in egg-positive semen samples than in egg-negative samples. Because SEA is a direct marker of the presence of eggs, the positive correlation between seminal SEA levels and egg counts seems logical.
Eggs in semen are associated with leukocytospermia and increased seminal levels of cytokines, and MGS has been hypothesized as a risk factor for HIV transmission in areas endemic for S. haematobium.6 Studies are urgently needed to elucidate this aspect of urogenital schistosomiasis. Furthermore, investigation of clinical manifestations of MGS is also warranted in the context of male reproductive health. ECP and SEA in seminal fluid, in addition to CAA in serum, may in this context represent important supplementary markers in assessment of MGS and associated morbidity. Both CAA in serum and urinary egg counts, which measure intensity of infection, showed a positive correlation with seminal egg counts. This observation is consistent with previous findings in a post-mortem study on S. haematobium-infected men.3 In that study, high egg counts in the bladder tissue were associated with a higher frequency and higher intensity of egg deposition in the genital organs. CAA showed the highest overall sensitivity, specificity and positive predictive value for egg excretion in urine and semen. Although seminal ECP and SEA reflects important aspects of MGS, i.e., egg-induced inflammation and egg deposition, their potential use seems limited from a clinical point of view. Obtaining semen samples is in general encumbered with skepticism because of cultural and ethical constraints. CAA and urinary egg counts constitute alternative and relevant indirect markers of MGS. Of these markers, urinary egg counts remain the cheapest and technically easiest indirect marker to use, particular from a public health perspective in low-resource areas.
Received June 22, 2007.
Accepted for publication June 25, 2008.
* Address correspondence to Peter D. C. Leutscher, DBL–Centre for Health Research and Development, Institute of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Jaegersborg Alle 1D, Charlottenlund/Copenhagen 2920, Denmark. E-mail: apd{at}sks.aaa.dk 
Authors’ addresses: Peter D. C. Leutscher, Claus M. Reimert, and Niels Ørnbjerg, DBL–Centre for Health Research and Development, Institute of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Jaegersborg Alle 1D, Charlottenlund/ Copenhagen 2920, Denmark, Tel: 45-77-32-77-32, Fax: 45-77-32-77-33, E-mail: apd{at}sks.aaa.dk. Govert T. J. van Dam and André M. Deelder, Department of Parasitology, Leiden University Medical Centre, Leiden, The Netherlands. Charles-Emile Ramarakoto, Department of Epidemiology, Institute Pasteur, Antananarivo, Madagascar.
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ABSTRACT
INTRODUCTION
