Maria Angeles Gómez-Morales

Validation of an Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Trichinellosis

ABSTRACT Trichinellosis is a zoonotic disease caused by the consumption of raw or semiraw meat from different animals harboring Trichinella larvae in their muscles. Since there are no pathognomonic signs, diagnosis can be difficult; for this reason, serology is important. The objective of this study was to validate an enzyme-linked immunosorbent assay (ELISA) using excretory/secretory antigens to detect anti-Trichinella immunoglobulin G antibodies in human sera. A total of 3,505 human serum samples were tested. A receiver-operator characteristic (ROC) curve analysis was performed. The accuracy of the test was determined by calculating the area under the curve, which was equal to 0.999, indicating high accuracy. The coefficient of variation calculated for data from four serum samples in eight working sessions was no higher than 5% for the positive sera or 14% for the negative sera. Moreover, the analysis of the differences in optical density between duplicates indicated a high repeatability for the ELISA. At the ROC optimized cutoff, the sensitivity and specificity of the test were, respectively, 99.2% and 90.6% (specificity of 95.6% when excluding the samples from multiparasitized persons from Tanzania). The validated ELISA showed good performance in terms of sensitivity, repeatability, and reproducibility, whereas the specificity was limited. These results suggest that this test is suitable for detecting anti-Trichinella antibodies in human sera for diagnostic purposes, whereas its use in epidemiological surveys could be questionable.

A distinctive Western blot pattern to recognize Trichinella infections in humans and pigs

Trichinellosis is a zoonotic disease caused by parasites of the genus Trichinella, which have a cosmopolitan distribution. For diagnostic purposes, a confirmatory test for ELISA-positive human and pig sera such as Western blotting is required, due to the high number of ELISA false positive sera. The objective of this study was to identify the Trichinella-specific antigens most frequently recognized by sera from Trichinella-infected humans and pigs, so as to define a distinctive pattern of Trichinella infection in sera from infected hosts using Western blots which allow false positive sera to be distinguished from true positive sera. Using excretory/secretory antigens, 450 human sera were tested by Western blotting: 150 from persons with a confirmed diagnosis of trichinellosis and 300 from persons who did not have trichinellosis but who tested positive by ELISA (i.e., false positives). We also tested 210 pig sera: (i) 30 from pigs experimentally infected with Trichinella spiralis; (ii) 90 from naturally T. spiralis-infected pigs; and (iii) 90 from pigs not infected with Trichinella, as shown after artificial digestion of the diaphragm pillars, yet which tested positive by ELISA (i.e., false positives). All true positive sera (i.e., sera from persons with confirmed trichinellosis as well as sera from naturally and experimentally infected pigs), reacted with a three-band pattern ranging in size from 48-72kDa. A distinctive pattern for recognizing Trichinella spp. infections in humans and pigs by Western blots is defined; it shows a sensitivity of 100% and it allows sera from Trichinella-infected humans and pigs to be distinguished from sera from persons and pigs that were not infected with Trichinella spp. (100% specificity).

Spatial distribution of Trichinella britovi, T. spiralis and T. pseudospiralis of domestic pigs and wild boars (Sus scrofa) in Hungary

Trichinellosis is a foodborne disease caused by the consumption of raw meat and raw meat-derived products from swine, horse and some game animals infected with nematode worms of the genus Trichinella. Between June 2006 and February 2011, 16 million domestic pigs and 0.22 million wild boars (Sus scrofa) were tested for Trichinella sp. in Hungary. Trichinella infection was not found in any pigs slaughtered for public consumption. Nevertheless, Trichinella spiralis was detected in four backyard pigs when trace back was done following a family outbreak. Trichinella infection was demonstrated in 17 wild boars (0.0077%). Larvae from wild boars were identified as Trichinella britovi (64.7%), T. spiralis (29.4%) and Trichinella pseudospiralis (5.9%). Although the prevalence of Trichinella sp. infection in wild boars and domestic pigs is very low, the spatial analysis reveals that the level of risk differs by region in Hungary. Most of the T. britovi infected wild boars (63.6%) were shot in the north-eastern mountain area of Hungary; whereas domestic pigs and wild boars infected with T. spiralis were detected only in the southern counties bordering Croatia and Romania. In the north-western and central counties, the prevalence of Trichinella infection seems to be negligible.

International ring trial to detect anti-Trichinella IgG by ELISA on pig sera.

To determine the reproducibility and robustness of an ELISA to detect anti-Trichinella IgG in pig sera which was previously validated at the Community Reference Laboratory for Parasites (CRLP), a ring trial was organized involving European and extra-European reference laboratories for Trichinella. The sensitivity and specificity of the assay determined by the CRLP validation resulted to be 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% and 96.9%, respectively. The analysis of the differences in optical density (OD) between duplicates indicated a high repeatability of the ELISA with about 95% of the differences between -0.16 and 0.17 absorbance units. The accuracy of the test was determined by calculating the area under the ROC curve (AUC). Overall, the ELISA index (I(E)) showed a very high accuracy (AUC=0.9965) and it performed significantly better than the mean of the duplicated ODs (AUC=0.9387). Of the 21 participating laboratories, nine performed the test without any modification of the original protocol, and 14 with some modifications. Of the laboratories that followed the protocol exactly, three produced false-negatives; whereas of the laboratories that modified the protocol, five produced false-negatives (differences between these two groups of laboratories were not significant, p=0.18). When comparing these two groups of laboratories, the AUCs were very similar (0.9988 and 0.9955, respectively). Finally, a normal mixed multiple model effect was used to evaluate if the I(E) obtained was only related to the serum or to other parameters such as the laboratory, dilution of the serum tested and application of the proposed protocol. The variability found in the test results was mainly due to the serum samples. The assay proposed is robust and reproducible and can be used for monitoring the lack of Trichinella infection in domestic pigs.

Patterns and risks of trichinella infection in humans and pigs in northern Laos.

Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory–secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI) = 17.1–21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.

Indirect versus direct detection methods of Trichinella spp. infection in wild boar (Sus scrofa).

BackgroundTrichinella spp. infections in wild boar (Sus scrofa), one of the main sources of human trichinellosis, continue to represent a public health problem. The detection of Trichinella spp. larvae in muscles of wild boar by digestion can prevent the occurrence of clinical trichinellosis in humans. However, the analytical sensitivity of digestion in the detection process is dependent on the quantity of tested muscle. Consequently, large quantities of muscle have to be digested to warrant surveillance programs, or more sensitive tests need to be employed. The use of indirect detection methods, such as the ELISA to detect Trichinella spp. infections in wild boar has limitations due to its low specificity. The aim of the study was to implement serological detection of anti-Trichinella spp. antibodies in meat juices from hunted wild boar for the surveillance of Trichinella spp. infections.MethodsTwo tests were used, ELISA for the initial screening test, and a specific and sensitive Western blot (Wb) as a confirmatory test. The circulation of anti-Trichinella IgG was determined in hunted wild boar muscle juice samples in 9 provinces of 5 Italian regions.ResultsFrom 1,462 muscle fluid samples, 315 (21.5%, 95% C.I. 19.51-23.73) were tested positive by ELISA. The 315 ELISA-positive muscle fluid samples were further tested by Wb and 32 (10.1%, 95% C.I. 7.29-13.99) of these were positive with a final seroprevalence of 2.2% (95% C.I 1.55-3.07; 32/1,462). Trichinella britovi larvae were detected by artificial digestion in muscle tissues of one (0.07%, 95%C.I. 0.01-0.39) out of the 1,462 hunted wild boars. No Trichinella spp. larvae were detected in Wb-negative wild boar. From 2006 to 2012, a prevalence of 0.017% was detected by muscle digestion in wild boar hunted in the whole Italian territory.ConclusionsThe combined use of both serological methods had a sensitivity 31.4 times higher than that of the digestion (32/1,462 versus 1/1,462), suggesting their potential use for the surveillance of the Trichinella spp. infection in wild boar populations.

Cryptic and Asymptomatic Opisthorchis felineus Infections

We describe the diagnostic difficulties experienced during an opisthorchiasis outbreak. Of 31 infected individuals, 61.3% were asymptomatic, and in the 12 symptomatic individuals, the duration of non-pathognomonic symptoms was shorter than 4 weeks. Serology by enzyme-linked immunosorbent assay and polymerase chain reaction fecal analysis were shown to be the most sensitive diagnostic tools.

Allergenic activity of Molicola horridus (Cestoda, Trypanorhyncha), a cosmopolitan fish parasite, in a mouse model

The cestode Molicola horridus is a muscle parasite of teleost fish. The ability of molecules present in this parasite to induce allergic response is not known yet. Since fish-borne parasitic allergens can induce allergic manifestations even when the parasitized fish is well cooked, the knowledge of potential allergens present in food is important in order to provide a save products for consumers. The aim of the study was to determine the allergenic potential of the components present in the crude larval extract (CLE) of M. horridus. Two mouse models were exposed to the CLE: adult BALB/c mice that were intraperitoneally (i.p.) immunized and newborn BALB/c mice that were orally exposed. Specific antibody levels in serum and faeces were measured by ELISA. The cellular immune response was determined by proliferation assay of splenocytes from sensitized mice. The protein profile of CLE was analysed by SDS-PAGE and western blot. In adult mice, specific IgG and IgA were detected in sera and faeces, whereas specific IgE were detected in sera only. In newborn mice, specific IgG were detected in sera and a low level of IgA was detected in faeces. SDS-PAGE revealed the CLE protein profile, with most of the proteins running from 15 to 50kDa. Specific IgG recognized mainly the 26 and 75kDa proteins and a molecular complex below 100kDa by immunoblot. Specific IgE recognized the same 26kDa protein as IgG did, and, with less intensity, another protein at 30kDa. Splenocytes from CLE-immunized mice proliferated when stimulated with CLE in a dose-dependent manner. The crude larval extract from M. horridus has potential allergenic molecules which can represent a risk for fish consumers.

Validation of an Excretory/Secretory Antigen Based-Elisa for the Diagnosis of Opisthorchis felineus Infection in Humans from Low Trematode Endemic Areas

Since opisthorchiasis does not show pathognomonic signs or symptoms, physicians can have serious problems to make a differential diagnosis of this infection in non endemic areas, in particular when there is a simultaneous occurrence with other seasonal infections. Moreover, symptomatic infections due to O. felineus can last a few weeks and then the signs and symptoms disappear, but the worms survive in the bile ducts for years causing hepatobiliary diseases including hepatomegaly, cholangitis, fibrosis of the periportal system, cholecystitis, and gallstones. Consequently, an early diagnosis prevents chronicity and loss of working days. The detection of specific antibodies has been considered as a complementary tool to the fecal examination to establish the definitive diagnosis of this infection and for the follow up. Therefore the aim of this work was the development and validation of an enzyme-linked immunosorbent assay (ELISA) using excretory/secretory antigens (ESA) from O. felineus adult worms to detect anti-Opisthorchis IgG in human sera. A total of 370 human sera were tested: 144 sera from persons with a confirmed diagnosis of opisthorchiasis, 110 sera from healthy Italian people, and 116 sera from people with other parasitic or non-parasitic infections. Results were analyzed by receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, calculated by the area under curve (AUC), yielded a 0.999 value, indicating the high performance of the test. The sensitivity was 100% (95% CI: 97.40% to 100%) and no false-negative sera were detected; the specificity was 99.09% (95% CI: 95.02% to 99.83%). The validated ELISA shows a good performance in terms of sensitivity, repeatability and reproducibility, and it is suitable to detect anti-Opisthorchis IgG in human sera for diagnostic purposes and for the follow up to assess the efficacy of drug treatment.

Epidemiology of taeniosis/cysticercosis in Europe, a systematic review: Western Europe

BackgroundTaenia solium and Taenia saginata are zoonotic parasites of public health importance. Data on their occurrence in humans and animals in western Europe are incomplete and fragmented. In this study, we aimed to update the current knowledge on the epidemiology of these parasites in this region.MethodsWe conducted a systematic review of scientific and grey literature published from 1990 to 2015 on the epidemiology of T. saginata and T. solium in humans and animals. Additionally, data about disease occurrence were actively sought by contacting local experts in the different countries.ResultsTaeniosis cases were found in twelve out of eighteen countries in western Europe. No cases were identified in Iceland, Ireland, Luxembourg, Norway, Sweden and Switzerland. For Denmark, Netherlands, Portugal, Slovenia, Spain and the UK, annual taeniosis cases were reported and the number of detected cases per year ranged between 1 and 114. Detected prevalences ranged from 0.05 to 0.27%, whereas estimated prevalences ranged from 0.02 to 0.67%. Most taeniosis cases were reported as Taenia spp. or T. saginata, although T. solium was reported in Denmark, France, Italy, Spain, Slovenia, Portugal and the UK. Human cysticercosis cases were reported in all western European countries except for Iceland, with the highest number originating from Portugal and Spain. Most human cysticercosis cases were suspected to have acquired the infection outside western Europe. Cases of T. solium in pigs were found in Austria and Portugal, but only the two cases from Portugal were confirmed with molecular methods. Germany, Spain and Slovenia reported porcine cysticercosis, but made no Taenia species distinction. Bovine cysticercosis was detected in all countries except for Iceland, with a prevalence based on meat inspection of 0.0002–7.82%.ConclusionsDetection and reporting of taeniosis in western Europe should be improved. The existence of T. solium tapeworm carriers, of suspected autochthonous cases of human cysticercosis and the lack of confirmation of porcine cysticercosis cases deserve further attention. Suspected cases of T. solium in pigs should be confirmed by molecular methods. Both taeniosis and human cysticercosis should be notifiable and surveillance in animals should be improved.