Field-based evaluation of a reagent strip test for diagnosis of schistosomiasis mansoni by detecting circulating cathodic antigen (CCA) in urine in low endemic area in Ethiopia

The sensitivity, specificity, positive and negative predictive values of a reagent strip test for the diagnosis of schistosomiasis mansoni by detecting circulating cathodic antigen (CCA) in urine were evaluated using 184 stool and urine samples collected from schoolchildren living in relatively low endemic area of schistosomiasis mansoni in Ethiopia. A combined result of stool samples processed by Kato and formol-ether concentration methods was used as gold standard. The results showed that detection of CCA in urine using reagent strip test was slightly higher than the combined results of the stool techniques (65.2 % vs 42.4 %, p > 0.05) in suggesting the prevalence of the disease. The sensitivity, specificity, positive and negative predictive values of the reagent strip test were 76.9 %, 43.4 %, 50 % and 71.9 %, respectively. The result of egg counts using Kato method suggested that detection of urine CCA could be used to indicate the intensity of infection. Nevertheless, like that of stool examination, the reagent strip test was found to be less sensitive in case of light to moderate infections. About 23.1 % of the study children who were excreting the eggs of the parasite were found negative by the reagent strip test. The relative insensitivity of a reagent strip test in low intensity of infection necessitates for the development of more sensitive assay that can truly discriminate schistosome-infected from non-infected individuals.     

Genitourinary schistosomiasis among pre-primary schoolchildren in a rural community within the Cross River Basin, Nigeria

In Africa, most schistosomiasis control programmes defined the age 5-19 years as the target population for nationwide control through the school systems, excluding the under fives. A study was therefore undertaken to determine the prevalence and intensity of genitourinary schistosomiasis in children aged 0-5 years (pre-primary) in Adim, a rural and endemic community within the Cross River Basin, Nigeria. Of the 126 children examined, 25 (19.8%) were infected with Schistosoma haematobium, with no significant difference (P > 0.05) in infection rates between boys (21.1%) and girls (18.2%). Both prevalence and intensity of infection increased significantly (P 0.05) between intensity in boys (6.2 eggs/10 ml urine) and girls (5.6 eggs/10 ml urine). A total of 32.5 and 27.8% of the children had haematuria and proteinuria, respectively; it was not gender specific (P > 0.05). Six species of snail were encountered, with Bulinus globosus being the most abundant and widespread. The results of this study have shown that pre-primary schoolchildren are a source of transmission of schistosomiasis in endemic communities and should be integrated into any control intervention.     

Schistosomiasis in Cross River State, Nigeria: 1. Prevalence and intensity of infection in Adim, Akamkpa Local Government Area

Mid-stream urine was randomly collected from 248 subjects in Adim. Blood and protein concentrations were determined semi-quantitatively using Combi-7 reagent strips. The urine samples were then processed and any ova of Schistosoma haematobium present were counted per 10 ml urine. Fresh stool samples were also randomly collected, processed and examined for S. masoni and other helminthic ova. The prevalence of S. haematobium in the area was 43.5% and this was found to be age-related but not sex-related. Mean egg count was 137.2 per 10 ml urine. Intense haematuria of 250 ery/microliters and proteinuria of 500 mg/dl accompanied the high egg counts. The stool examination showed no cases of Manson's schistosomiasis but polyparasitism with other intestinal helminths was common particularly among children under 10 years old. This is the first report of urinary schistosomiasis in this area and the high prevalence rate is consistent with the rice farming occupation of the natives of the area. The sensitive nature and the case of application of the reagent strips in determining heavy infections by measuring haematuria and proteinuria is once again confirmed.     

Infection intensity-dependent accuracy of reagent strip for the diagnosis of Schistosoma haematobium and estimation of treatment prevalence thresholds

BackgroundReagent strip to detect microhematuria as a proxy for Schistosoma haematobium infections has been considered an alternative to urine filtration for individual diagnosis and community-based estimates of treatment needs for preventive chemotherapy. However, the diagnostic accuracy of reagent strip needs further investigation, particularly at low infection intensity levels.MethodsWe used existing data from a study conducted in Tanzania that employed urine filtration and reagent strip testing for S. haematobium in two villages, including a baseline and six follow-up surveys after praziquantel treatment representing a wide range of infection prevalence. We developed a Bayesian model linking individual S. haematobium egg count data based on urine filtration to reagent strip binary test results available on multiple days and estimated the relation between infection intensity and sensitivity of reagent strip. Furthermore, we simulated data from 3,000 hypothetical populations with varying mean infection intensity to infer on the relation between prevalence observed by urine filtration and the interpretation of reagent strip readings.Principal findingsReagent strip showed excellent sensitivity even for single measurement reaching 100% at around 15 eggs of S. haematobium per 10 ml of urine when traces on reagent strip were considered positive. The corresponding specificity was 97%. When traces were considered negative, the diagnostic accuracy of the reagent strip was equivalent to urine filtration data obtained on a single day. A 10% and 50% urine filtration prevalence based on a single day sampling corresponds to 11.2% and 48.6% prevalence by reagent strip, respectively, when traces were considered negative, and 17.6% and 57.7%, respectively, when traces were considered positive.Conclusions/SignificanceTrace results should be included in reagent strip readings when high sensitivity is required, but excluded when high specificity is needed. The observed prevalence of reagent strip results, when traces are considered negative, is a good proxy for prevalence estimates of S. haematobium infection by urine filtration on a single day.     

Evaluation of urine CCA assays for detection of Schistosoma mansoni infection in Western Kenya

Although accurate assessment of the prevalence of Schistosoma mansoni is important for the design and evaluation of control programs, the most widely used tools for diagnosis are limited by suboptimal sensitivity, slow turn-around-time, or inability to distinguish current from former infections. Recently, two tests that detect circulating cathodic antigen (CCA) in urine of patients with schistosomiasis became commercially available. As part of a larger study on schistosomiasis prevalence in young children, we evaluated the performance and diagnostic accuracy of these tests--the carbon test strip designed for use in the laboratory and the cassette format test intended for field use. In comparison to 6 Kato-Katz exams, the carbon and cassette CCA tests had sensitivities of 88.4% and 94.2% and specificities of 70.9% and 59.4%, respectively. However, because of the known limitations of the Kato-Katz assay, we also utilized latent class analysis (LCA) incorporating the CCA, Kato-Katz, and schistosome-specific antibody results to determine their sensitivities and specificities. The laboratory-based CCA test had a sensitivity of 91.7% and a specificity of 89.4% by LCA while the cassette test had a sensitivity of 96.3% and a specificity of 74.7%. The intensity of the reaction in both urine CCA tests reflected stool egg burden and their performance was not affected by the presence of soil transmitted helminth infections. Our results suggest that urine-based assays for CCA may be valuable in screening for S. mansoni infections.     

Evaluation of Circulating Cathodic Antigen (CCA) Urine-Tests for Diagnosis of Schistosoma mansoni Infection in Cameroon

Background

The Kato-Katz is the most common diagnostic method for Schistosoma mansoni infection. However, the day-to-day variability in host egg-excretion and its low detection sensitivity are major limits for its use in low transmission zones and after widespread chemotherapy. We evaluated the accuracy of circulating cathodic antigen (CCA) urine-assay as a diagnostic tool of S. mansoni. In comparison, a low sensitive CCA test (CCA-L) was assessed.

Methodology

The study was conducted in three settings: two foci with single S. mansoni infections (settings A and B), and one mixed S. mansoni – S. haematobium focus (setting C). Stool and urine samples were collected from school-children on three consecutive days. Triplicate Kato-Katz readings were performed per stool sample. Each urine sample was tested with one CCA and only the first urine sample was subjected to CCA-L. Urine samples were also examined for S. haematobium eggs using the filtration method and for microhaematuria using urine reagent strips. Overall, 625 children provided three stool and three urine samples.

Principal Findings

Considering nine Kato-Katz thick smears as ‘reference’ diagnostic test, the prevalence of S. mansoni was 36.2%, 71.8% and 64.0% in settings A, B and C, respectively. The prevalence of S. haematobium in setting C was 12.0%. The sensitivities of single Kato-Katz, CCA and CCA-L from the first stool or urine samples were 58%, 82% and 46% in setting A, 56.8%, 82.4% and 68.8% in setting B, and 49.0%, 87.7% and 55.5% in setting C. The respective specificities were 100%, 64.7% and 100%; 100%, 62.3% and 91.3%; and 100%, 42.5% and 92.0%. Mixed infection with S. haematobium did not influence the CCA test results for S. mansoni diagnosis.

Conclusions/Significance

Urine CCA revealed higher sensitivity than CCA-L and triplicate Kato-Katz, and produced similar prevalence as nine Kato-Katz. It seems an attractive method for S. mansoni diagnosis.     

Schistosomiasis at Loum, Cameroun; Schistosoma haematobium, S. intercalatum and their natural hybrid.

A survey of 500 schoolchildren in Loum in 1968 revealed an overall infection rate of 54.2% with Schistosoma intercalatum and this was the only species of schistosome encountered. In 1972 a number of children were found to be passing schistosome eggs in their urine and these eggs ranged in shape and size from the forms characteristic for S. haematobium to those of S. intercalatum. Preliminary laboratory studies demonstrated that hybridisation between the two species was occurring. Subsequent field surveys showed that the snail hosts for the two parasites (B. rohlfsi for S. haematobium and B. forskali for S. intercalatum) were both present in the river Mbette and its tributaries in Loum and the distribution of the two snail species coincided closely with the distribution of the schistosomes in the human population. Detailed study of a small group of children passing hybrid eggs in their urine revealed that few of them were passing eggs in their faeces and that those eggs which were found in faeces were not viable. Analysis of schistosome egg-shape by plotting cumulative size-frequency data on probability paper demonstrated that the graph obtained from a natural hybrid series was different from that given by a known mixture of the two separate species. The hybrid series included a number of exceptionally large eggs resembling those of S. bovis but isolation of these eggs and subsequent laboratory passage of the parasites showed that they were part of the series and were not evidence of the presence of a third species. Hybridisation experiments in the laboratory showed that the cross S. haematobium male X S. intercalatum femal is fully viable but that the reverse mating is not successful, thus accounting for the failure of the faecal eggs recovered from children with hybrid infections. Histological results from laboratory passaged hybrids suggest that the Ziehl-positive staining reaction of the egg-shells of S. intercalatum may be a recessive character. The observations reported here indicate that S. haematobium has only recently become established in Loum and that it is, through introgressive hybridisation, replacing the indigenous S. intercalatum. A suggested explanation for the change in the parasite fauna is offered and this depends upon ecological changes resulting from forest clearance and agricultural development providing improved conditions for the spread of B. rohlfsi, the snail host for S. haematobium. It is suggested that, in contrast to recent reports on the spread of S. intercalatum, this species is in fact retreating and being replaced by S. haematobium in areas where forest clearance is taking place. In conclusion it is suggested that introgressive hybridisation of this kind may have been responsible for the evolution of certain characteristic local strains of African schistosomes.     

Field assessment in Cameroon of a reader of POC-CCA lateral flow strips for the quantification of Schistosoma mansoni circulating cathodic antigen in urine

BackgroundDetermining Schistosoma mansoni infection rate and intensity is challenging due to the low sensitivity of the Kato-Katz (KK) test that underestimates the true disease prevalence. Circulating cathodic antigen (CCA) excreted in urine is constantly produced by adult worms and has been used as the basis of a simple, non-invasive point of care test (POC-CCA) for Schistosoma mansoni infections. Although the abundance of CCA in urine is proportional to worm burden, the POC-CCA test is marketed as a qualitative test, making it difficult to investigate the wide range of infection intensities. This study was designed to compare the prevalence and intensity of S. mansoni by KK and POC-CCA and quantify, on fresh and frozen (<-20°C) urine samples, CCA using the visual scores and the ESEquant LR3 reader.MethodologyStool and urine samples were collected from 759 school-aged children. The prevalence and intensity of S. mansoni were determined using KK and POC-CCA. The degree of the positivity of POC-CCA was estimated by quantifying CCA on fresh and frozen urine samples using visual scores and strip reader. The prevalence, the infection intensity as well the relative amounts of CCA were compared.ResultsThe S. mansoni infection rates inferred from POC-CCA and KK were 40.7% and 9.4% respectively. Good correlations were observed between infection intensities recorded by; i) the reader and visual scoring system on fresh (Rho = 0.89) and frozen samples (Rho = 0.97), ii) the reader on fresh urine samples and KK (epg) (Rho = 0.44). Nevertheless, 238 POC-CCA positive children were negative for KK, and sixteen of them had high levels of CCA. The correlation between results from the reader on fresh and frozen samples was good (Rho = 0.85). On frozen samples, CCA was not detected in 55 samples that were positive in fresh urine samples.ConclusionThis study confirmed the low sensitivity of KK and the high capacity of POC-CCA to provide reliable data on the prevalence and intensity of S. mansoni infections. The lateral flow reader enabled accurate quantification of CCA under field conditions on fresh and frozen urine samples with less time and effort than KK.     

Schistosoma haematobium infections in preschool children from two rural communities in Ijebu East, south-western Nigeria

There is an urgent need for information on schistosomiasis in preschool children, who are often excluded in mass treatment programmes. The prevalence and intensity of Schistosoma haematobium infection were determined in preschool children aged ≤ 6 years in two rural communities in Ijebu East, south-western Nigeria. Two urine samples each were collected from 83 preschool children from the two communities, tested for microhaematuria using reagent strips and then processed and examined with a microscope for S. haematobium eggs. Focus group discussions on perceptions of the disease and water contact practices were held in the communities with their guardians, caregivers and preschool children, using an interview guide. The prevalence of S. haematobium in the two communities was 14 (16.9%), with no significant differences (P = 0.661) in infection rate between boys (18.4%) and girls (14.7%). Both prevalence and intensity of infection did not increase significantly with age in both Korede and Obada community. However, there were significant differences in prevalence of infection between the two communities (P = 0.035). There was no association (P = 0.750) between intensity in boys (0.176 eggs/10 ml urine) and girls (0.110 eggs/10 ml urine). Focal group discussions with guardians and caregivers revealed that preschool children acquired infection early in their lives through exposure to infected stream water by their mothers, while the older children visit the stream for playing, bathing and swimming. It has therefore become imperative for preschool children to be included in the planning of schistosomiasis intervention programmes as a means of reducing transmission.     

Urinary schistosomiasis in southern Ghana: 1. Prevalence and morbidity assessment in three (defined) rural areas drained by the Densu river

Epidemiological studies on urinary schistosomiasis were carried out in eight villages in the Ga and Akuapem South districts in Ghana. Single urine samples were collected from individuals aged 5 years and above between 10.00 and 14.00 h. The samples were examined for the presence of Schistosoma haematobium eggs using a filtration technique. Indirect morbidity was determined as the presence of microhaematuria and proteinuria using reagent strips, and macrohaematuria was recorded with the naked eye. Out of the study population of 3912 subjects, 2562 (65.5%) submitted urine samples. The prevalence of a Schistosoma haematobium infection ranged between 54.8 and 60.0%. Infection rates increased by age with a peak in the 10-19 years category, and decreased with increasing age. Disease prevalence was higher in males aged 15 years and above in Areas 2 (Ntoaso and Sansami Amanfro) and 3 (Dom Faase, Papase, Chento and Gidi Kope), whereas it was higher among males aged 10 years and above in Area 1 (Ayikai Doblo and Akramaman). The intensity of infection was highest among children aged 10-14 years in most of the villages. More than half of egg-positive children in this age group had a heavy infection (100 eggs and above in 10 ml of urine). Although both egg-positive and egg-negative individuals manifested variable degrees of macro- or micro-haematuria, microhaematuria was more prevalent among egg-positives (chi(2)=918.5, d.f.=1, P<0.01). The degree of microhaematuria and proteinuria were significantly associated with the intensity of the infection. These results indicate a high transmission of disease in the study area.     

Screening trematodes for novel intervention targets: a proteomic and immunological comparison of Schistosoma haematobium, Schistosoma bovis and Echinostoma caproni

With the current paucity of vaccine targets for parasitic diseases, particularly those in childhood, the aim of this study was to compare protein expression and immune cross-reactivity between the trematodes Schistosoma haematobium, S. bovis and Echinostoma caproni in the hope of identifying novel intervention targets. Native adult parasite proteins were separated by 2-dimensional gel electrophoresis and identified through electrospray ionisation tandem mass spectrometry to produce a reference gel. Proteins from differential gel electrophoresis analyses of the three parasite proteomes were compared and screened against sera from hamsters infected with S. haematobium and E. caproni following 2-dimensional Western blotting. Differential protein expression between the three species was observed with circa 5% of proteins from S. haematobium showing expression up-regulation compared to the other two species. There was 91% similarity between the proteomes of the two Schistosoma species and 81% and 78·6% similarity between S. haematobium and S. bovis versus E. caproni, respectively. Although there were some common cross-species antigens, species-species targets were revealed which, despite evolutionary homology, could be due to phenotypic plasticity arising from different host-parasite relationships. Nevertheless, this approach helps to identify novel intervention targets which could be used as broad-spectrum candidates for future use in human and veterinary vaccines.     

Transmission of urinary schistosomiasis in Sukumaland, Tanzania. 1. Snail infection rates and incidence of infection in school children

Seasonal density fluctuations of Bulinus nasutus populations with accompanying Schistosoma haematobium infection rates in relation to rainfall and habitat water volumes were studied at Ukiriguru, Mwanza, Tanzania. Alongside the snail investigations, 50 school children initially negative for urinary schistosomiasis were examined regularly to determine seasonal incidence rates for the infection. Of the 17,646 B. nasutus collected in 2 years, 156 (0.88%) were found shedding cercariae. Snail populations fluctuated seasonally as influenced by rainfall through its effects on habitat water volume. Monthly snail infection rates ranged from 0.09% to 3.19% and were highest in February and March, at the time of the short dry period. Monthly incidence of S. haematobium in school children ranged between 2.6% and 12.5%, being highest in April and May. There was a significant linear association between monthly snail infection rates and S. haematobium incidence rates in school children (r2s = 0.65 in 1982/83; r2s = 0.87 in 1983/84) suggesting that the maximum transmission period for urinary schistosomiasis in the area occurs during the short dry period, sometime in February/March so that most of the infections in the community would be detected in April/May.     

Endemicity, focality and seasonality of transmission of human schistosomiasis in Amagunze Village, eastern Nigeria

The pattern of transmission of human schistosomiasis was studied in Amagunze Village, eastern Nigeria, during 1986-1987. The prevalence of Schistosoma haematobium in 119 schoolboys aged 5-12 years was 79%. The geometric mean of intensity of infection was 49 eggs/10 ml urine and the frequency of visible haematuria was 25.2%. No S. mansoni infections were demonstrated. A marked seasonality in population density of Bulinus truncatus, B. forskalii and Biomphalaria pfeifferi was demonstrated with reduced densities during the late rainy and early dry seasons. Schistosoma sp. infected B. truncatus were found in the late dry and early rainy seasons in 2 out of 7 major human water contact sites studied. Seasonality and focality of transmission of S. haematobium and its high endemicity in the area were thus demonstrated.     

Urinary schistosomiasis on Zanzibar: application of two novel assays for the detection of excreted albumin and haemoglobin in urine

As part of a urinary schistosomiasis control programme on Zanzibar, an aged cross-sectional survey of 305 children from three schools on Unguja was conducted to investigate the relationships between levels of excreted albumin and haemoglobin in urine and Schistosoma haematobium infection status. Diagnosis was determined by standard parasitological methods, dipstick reagents for microhaematuria, visual inspection for macrohaematuria as well as collection of case-history questionnaire data for self-diagnosis. Prevalence of infection as determined by parasitology was 53.9% and approximately, one quarter of the children examined were anaemic (<11 g dl(-1)). A statistically significant negative association of blood haemoglobin levels of boys and S. haematobium infection intensity status was observed (rs=-0.23, P=0.005). Through sensitivity analysis of urine-albumin values it was determined that a concentration of above >40 mg l(-1), as measured with the HemoCue urine-albumin photometer, had sensitivity, specificity, positive and negative predictive values of 0.90, 0.83, 0.86 and 0.89 respectively against 'gold-standard' parasitology. There was a clear association of reported pain upon micturition for children with elevated urine-albumin levels, with an odds ratio of 20 to 1. Levels of excreted blood in urine were quantified with the HemoCue Plasma/Low Hb photometer. However, dipsticks remain the method of choice for urine-haemoglobin of 0.1 g l(-1) and below. Urine parameters over a 24-h period were assessed in a small sub-sample. Reductions in both albumin and haemoglobin excretion were observed in 11 children 54 days after praziquantel treatment. It was concluded that these rapid, high-through-put, portable HemoCue assays could play a role in better describing and monitoring the occurrence, severity and evolution of urinary schistosomiasis disease. The urine-albumin assay has particular promise as a biochemical marker of S. haematobium induced kidney- and upper urinary tract-morbidity.     

Seasonal patterns in the transmission of Schistosoma haematobium in Attaouia, Morocco.

In the Attaouia area, the density of Bulinus truncatus (Audouin, 1827), was monitored monthly for a period of one year in correlation with weather variations. Snails were active throughout the year and particularly abundant at the end of spring and summer. Two snail generations were found to overlap. The infection rate of B. truncatus reached a maximum of 3.5% in the summer when human water contact was frequent. A selective survey conducted in the village of Lamyayha showed that the prevalence of infection with S. haematobium among the local population was 21.2% who were passing from 10 to 80 eggs per 10 ml of urine.     

Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.     

Single dose oral treatment in urinary schistosomiasis: a double blind trial.

A double blind trial of three oral preparations given in single doses for the treatment of Schistosoma haematobium infection was carried out in schoolchildren; selection was biased towards those who excreted large quantities of eggs. Praziquantel 40 mg/kg was the most effective drug giving a greater than 97% reduction in egg output six months after treatment; combined treatment with niridazole 25 mg/kg and metrifonate 10 mg/kg gave a reduction of greater than 92% and metrifonate 10 mg/kg alone a reduction of greater than 86%. Fewer children continued to have moderate to heavy infections (excretion greater than 124 ova/10 ml urine) six months after treatment with praziquantel (5%) and the combined regimen (7%) than with metrifonate (16%). Though our findings show that praziquantel appears to be the most effective and convenient drug available for individuals with S haematobium infection, the combined regimen is a cheaper alternative for treatment where cost is important and parasitological cure not an essential objective.     

Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse,hamster, and human infections

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.