Jose R. Espinoza

Immunodiagnosis of human fascioliasis with Fasciola hepatica cysteine proteinases

The immunodiagnosis of human infections with Fasciola hepatica using purified parasite cysteine proteinases as antigens is reported in this paper. IgG enzyme-linked immunosorbent assays (ELISAs) with 2 cysteine proteinases of 26 kDa (Fas1) and 25 kDa (Fas2), obtained from the regurgitated material of adult worms, were evaluated with serum samples from 38 patients infected with F. hepatica, from 54 patients with other parasitic infections and from 46 healthy volunteers. The diagnostic sensitivities for detection of F. hepatica infection at a serum dilution of 1 in 500 were 89% and 95% with Fas1 and Fas2 respectively. The specificity of the ELISAs was 98% for Fas1 and 100% for Fas2. IgG ELISA with Fas2 is a highly sensitive and specific assay for the immunodiagnosis of human fascioliasis.

Fasciolosis humana y animal en el Perú: impacto en la economía de las zonas endémicas

Fasciola hepatica is the causative agent of fasciolosis in Peru; the disease is an important public health problem by the high prevalence of the human infection affecting mainly children and a major veterinary problem by the high rates of infected livestock. The human disease is endemic in the Sierra and the Coast but sporadic in the Amazonia, and reported in 18 Departments, while the animal infection in 21 of 24 Departments of Peru. Transmission occurs in Andean rural populations engaged in agriculture, but recently an increasing number of people became infected in the cities. The epidemiological situation in Peru includes i) Departments with non-autochtonous cases, where infection occurs by consumption of contaminated vegetables brought from endemic areas or infection is acquired by visit to endemic areas; ii) Departments with hypoendemic and mesoendemic villages, where transmission occurs by ingest of contaminated vegetables and prevalence ≤10%; and iii) Departments with hyper-endemic villages with human prevalence >10 %, with an intense transmission by consumption of contaminated vegetables. The disease affects bovine, sheep, goat, swine, equine, South American camelids, rabbits and guinea pigs. The negative impact of fasciolosis in the livestock economy is not lesser than US

Fas2-ELISA in the detection of human infection by Fasciola hepatica.

50 million per year, estimation based on the prevalence and the number of condemned livers in the abattoirs. It is difficult to estimate the economic impact of this infection in the human health due to its status of neglected disease, but fasciolosis is hyper-endemic in the poorest Andean areas of Peru where the situation has to be recognized as a public health emergency.

Genetic susceptibility to different clinical forms of tuberculosis in the Peruvian population

Fasciola hepatica has recently emerged as a major pathogen of humans from reports on areas of endemicity and hyper-endemicity for fascioliasis. This situation is aggravated by the lack of standard assays for the screen diagnosis of F. hepatica infection in humans living in endemic areas. Our laboratory has developed an enzyme-linked immunosorbent assay (Fas2-ELISA) based on the capture of IgG antibody by a purified protein Fas2, which is an adult fluke cysteine proteinase. Fas2-ELISA exhibited 95% sensitivity and 100% specificity in 38 individuals infected with F. hepatica diagnosed by finding eggs in stools and 46 serum samples from healthy volunteers. No cross-reaction was observed with 54 serum samples from patients with ten different parasitic infections including the trematodes Paragonimus westermani and Schistosoma mansoni. The high antigenicity of Fas2 is suggested by the fact that antibodies to Fas2 rise rapidly by 1-2 weeks of infection and rise until patency at 8 weeks of infection in experimentally infected alpacas. Field screening for human fascioliasis using Fas2-ELISA and coprology in three endemic locations of the Peruvian Andes resulted in 95.5% sensitivity, 86.6% specificity in a population of 664 children in an age range of 1 to 16 years old. These results provide evidence of the clinical potential of Fas2-ELISA to diagnose fascioliasis in humans exposed to liver fluke infection in endemic areas for this parasite. Fas2-ELISA is currently developed as a standard assay for both field screening for fascioliasis in people living in endemic areas and detecting occasionally F. hepatica infected patients in clinical laboratories.

Fascioliasis in relatives of patients with Fasciola hepatica infection in Peru

Racial variation, twin studies, segregation analyses, linkage and association studies all suggest that genetic factors play an important role in predisposition to tuberculosis. Many previous studies have been performed with pulmonary TB patients, as the most prevalent form of clinical TB (nearly 95%), and very few of them have considered extrapulmonary TB. The present study evaluates the effects of variation in eight candidate genes (LTA, TNF, IL1B, IL1RN, IL10, TGFB1, TIRAP and P2X7) with pulmonary, pleural, miliary and other extrapulmonary forms of TB in a Peruvian population from the North of Lima. 626 TB cases and 513 healthy controls were enrolled in this study. LTA(+368) and IL10(-592) were associated with different clinical forms of TB (P<0.05). LTA(+368) genotype A/A was protective for pleural TB, LTA(+368) G/A was correlated with susceptibility to miliary TB. Genotypes A/A and G/A were associated with protection and susceptibility respectively when considering all extrapulmonary TB forms versus either healthy controls or pulmonary TB patients. Carriers of IL10(-592)*C were under-represented among those with pulmonary TB and all TB forms (P<0.001). IL10(-1082)-IL10(-592) haplotypes showed different distributions among patients with pulmonary TB and all TB forms (P<0.01) when compared to healthy controls. In addition, IL10(-1082)-IL10(-592) haplotypes showed differences between pleural, miliary and all forms of extrapulmonary TB when compared with pulmonary TB (P<0.05). All findings are consistent with an under-representation of the IL10(-1082)*A-IL10(-592)*A haplotype in pulmonary TB patients. These results suggest that the polymorphisms LTA(+368) and IL10(-592), or variants in strong linkage disequilibrium, variably affect susceptibility to the differing clinical forms of TB in Peruvians.

Fasciola hepatica phenotypic characterization in Andean human endemic areas: Valley versus altiplanic patterns analysed in liver flukes from sheep from Cajamarca and Mantaro, Peru

High prevalence rates of human fascioliasis have been described in several regions of Peru. We surveyed 20 families in an endemic area of Peru in order to determine the proportion of infection with F. hepatica in relatives of diagnosed subjects and in order to identify associated risk factors. The study included feces and blood samples of 93 subjects. Ages ranged from one to 53 (mean = 18.6; SD = 14.2). The overall prevalence of fascioliasis by fecal examinations was 33.3% (n = 83) and by serology, 51.9% (n = 86). The prevalence in age group I (< or = 19 years old) by coprological and serological tests was 61.4% and 75.9%, respectively; in group II (> 19 years old) 15.4% and 37.5%. The main associated risk factor with fascioliasis was eating salads (OR = 3.29, CI = 1.2 - 9.0, p = 0.02). In conclusion, human fascioliasis is highly prevalent in the relatives of index cases and the most significant risk factor of acquiring fascioliasis in the family is eating salads in endemic areas.

Field Evaluation of a Coproantigen Detection Test for Fascioliasis Diagnosis and Surveillance in Human Hyperendemic Areas of Andean Countries

Fascioliasis is a zoonotic parasitic disease caused by Fasciola hepatica and Fasciola gigantica. Of both species, F. hepatica is the only one described in the Americas, mainly transmitted by lymnaeid snail vectors of the Galba/Fossaria group. Human fascioliasis endemic areas are mainly located in high altitude areas of Andean countries. Given the necessity to characterize F. hepatica populations involved, the phenotypic features of fasciolid adults infecting sheep present in human fascioliasis endemic areas were analysed in the Cajamarca Valley and Mantaro Valley (valley transmission patterns) and the northern Bolivian Altiplano (altiplanic transmission pattern). A computer image analysis system (CIAS) was applied on the basis of standardized measurements. The aforementioned highland populations were compared to standard lowland natural and experimental populations of European origin. Liver fluke size was studied by multivariate analyses. Two phenotypic patterns could be distinguished in F. hepatica adult size: the valley pattern (Cajamarca and Mantaro, Peru) and the altiplanic pattern (northern Altiplano, Bolivia). Results showed that the Andean valley population and European standard populations presented a phenotypic homogeneity. The Altiplano population showed a large size range with a pronouncedly lower minimum size indicating that uterus gravidity is reached at a smaller size than in valley populations. The results of this study demonstrate that there is no apparent relationship between the shape of fasciolid adults with regard to altitudinal difference or geographical origin and that allometry-free shape appears as a more stable trait than size in fasciolid species. Results are analysed in terms of intensity/crowding effect aspects and permanent/seasonal transmission characteristics.

Cysteine proteinases Fas1 and Fas2 are diagnostic markers for Fasciola hepatica infection in alpacas (Lama pacos)

Background Emergence of human fascioliasis prompted a worldwide control initiative including a pilot study in a few countries. Two hyperendemic areas were chosen: Huacullani, Northern Altiplano, Bolivia, representing the Altiplanic transmission pattern with high prevalences and intensities; Cajamarca valley, Peru, representing the valley pattern with high prevalences but low intensities. Coprological sample collection, transport and study procedures were analyzed to improve individual diagnosis and subsequent treatments and surveillance activities. Therefore, a coproantigen-detection technique (MM3-COPRO ELISA) was evaluated, using classical techniques for egg detection for comparison. Methodology and Findings A total of 436 and 362 stool samples from schoolchildren of Huacullani and Cajamarca, respectively, were used. Positive samples from Huacullani were 24.77% using the MM3-COPRO technique, and 21.56% using Kato-Katz. Positive samples from Cajamarca were 11.05% using MM3-COPRO, and 5.24% using rapid sedimentation and Kato-Katz. In Huacullani, using Kato-Katz as gold standard, sensitivity and specificity were 94.68% and 98.48%, respectively, and using Kato-Katz and COPRO-ELISA test together, they were 95.68% and 100%. In Cajamarca, using rapid sedimentation and Kato-Katz together, results were 94.73% and 93.58%, and using rapid sedimentation, Kato-Katz and copro-ELISA together, they were 97.56% and 100%, respectively. There was no correlation between coproantigen detection by optical density (OD) and infection intensity by eggs per gram of feces (epg) in Cajamarca low burden cases (<400 epg), nor in Huacullani high burden cases (≥400 epg), although there was in Huacullani low burden cases (<400 epg). Six cases of egg emission appeared negative by MM3-COPRO, including one with a high egg count (1248 epg). Conclusions The coproantigen-detection test allows for high sensitivity and specificity, fast large mass screening capacity, detection in the chronic phase, early detection of treatment failure or reinfection in post-treated subjects, and usefulness in surveillance programs. However, this technique falls short when evaluating the fluke burden on its own.

Detection of antibodies against Fasciola hepatica in cirrhotic patients from Peru

Circulating antibody against Fasciola hepatica antigens was determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis in alpacas naturally exposed to F. hepatica. Serological assay parameters were established by using sera from eight infected animals and seven controls with no record of this parasitic infection. Excretory--secretory (ES-) products, Fas1- and Fas2-ELISA were used to survey 307 alpacas from a F. hepatica endemic area in the Peruvian Andes. Seroprevalence of F. hepatica infection varied from 56.7, 64.8 and 66.8% measured by Fas1-, Fas2- and ES-ELISA, respectively. The sensitivity for ES-ELISA was 95%, corresponding Fas1- and Fas2-ELISA sensitivity values were 90 and 95%. In this population, 7% of animals were positive for F. hepatica eggs in faeces, other parasites detected were Trichuris sp. (40%), Nematodirus sp. (34.6%), Lamanema sp. (12.8%) and Eimeria sp. (11.8%). The results show that F. hepatica infected animals elicit circulating antibodies against ES, Fas1 and Fas2. Fas2-ELISA may be proposed as a sensitive assay for the immunodiagnosis of fasciolosis in alpacas.

Mechanisms of Liver Fibrosis Associated with Experimental Fasciola hepatica Infection: Roles of Fas2 Proteinase and Hepatic Stellate Cell Activation

The prevalence of Fasciola hepatica infection, in endemic countries, in patients with established cirrhosis is unknown. We hypothesized that, in endemic countries, the presence of fascioliasis may be detected in a serum pool of cirrhotic patients. Forty-four previously stored serum samples of patients with established liver cirrhosis, in the Hospital Nacional Cayetano Heredia in Lima, Peru, were collected from 1998 to 2003 and assessed for hepatitis B, C and fascioliasis antibodies (Fas2 ELISA). Hepatitis B surface antigen (HBsAg) was positive in 8.8% (n = 34), hepatitis B core antibody (anti-HBc) in 32.5% (n = 34), hepatitis C antibodies (anti-HCV) in 9.1% (n = 33), and 9.1% (n = 44) were Fas2 ELISA positive. This disease is an example of an emerging tropical infection which can be present in chronic liver diseases, requiring greater clinician awareness especially in endemic rural areas. Further clinical studies are warranted.