Molecular based confirmation of puma meat sausages implicated in trichinellosis outbreaks in Argentina.

Trichinellosis is a zoonotic disease caused by Trichinella, with the main source of infection being the consumption of pork and pork-derived products. However, it can also be acquired from eating the meat from wild animals targeted for sport hunting. The objectives of this study were: 1) to develop and implement a molecular method for the identification of Sus scrofa (pig and wild boar) and Puma concolor (Puma) meat in sausages eaten raw, which were linked to trichinellosis outbreaks occurring in Córdoba, Buenos Aires and La Pampa provinces, Argentina, in 2010, 2021, and 2022, respectively; and 2) to identify the Trichinella species present in the food. Specific primers were designed for PCR amplification and nucleotide sequencing of a region of the mitochondrial cytochrome b gene from both host species. Samples from the mentioned outbreaks were analysed, and the molecular identification of Trichinella spp. larvae was also performed. Results of the species identification system revealed that sausages from Córdoba and Buenos Aires had a mixed composition of pork and puma meat, while those from La Pampa contained puma meat only. Trichinella spiralis was implicated in all three outbreaks. The species identification system developed and implemented in this study revealed two host species of Trichinella related to human cases, and alerts about the risk of zoonotic transmission to humans through infected puma meat.

A review on Trichinella infection in South America.

Trichinella spp. causes human trichinellosis by means of the consumption of raw or inadequately treated meat from domestic or game animals. In the Americas, as well as in other continents, Trichinella infection is a health issue for humans and has a negative impact on the pork meat market, generated by people's fear of becoming infected with the parasite. The distribution of human cases and the sources of this disease in humans and animals were analysed in this report, which summarizes the information available regarding Trichinella infection in animals and humans in South America. Within South America, human infection with Trichinella was documented in Argentina and Chile during the period 2005-2019. Trichinellosis is endemic in these countries for, with human cases and foci in domestic and wild animals. In Argentina, human cases occur throughout the country, with foci found in pigs and wild animals. In Argentina, during the period 2012-2018, the number of suspected human cases reached 6,662. T. spiralis was identified in one South American sea lion (Otaria flavescens) from Patagonia, Argentina, for the first time in the region in 2018. In Chile, 258 human cases of trichinellosis were confirmed during the period 2005-2015; out of those 258 cases, most samples which tested positive for Trichinella spp. (29.5%) were detected in the Metropolitan district (Santiago de Chile and outskirts), and 17.4% in The Lake district. Regarding age brackets, people between 30-49 years of age showed the most cases (40.1%). In Brazil, the infection is absent in domestic species but it has been found in wild boars (Sus scrofa) but limited to one or more region of the country. Within the animal species destined for food in South America, those that showed higher parasitical loads were pigs and wild boars, while armadillos (Chaetophractus villosus) and peccaries (Tayassu tajacu) showed very low Trichinella spp. larvae loads (0.04 - 0.1 larvae/g). Antibodies against Trichinella spp. have been detected in pigs from Ecuador and Bolivia. In Bolivia, antibodies were also found in humans. Peru, Colombia and Uruguay have no documented presence of Trichinella spp. in animals and humans. There is insufficient information regarding the presence of Trichinella spp. in domestic and wild animals, as well as in humans, since only a very limited number of surveys have been carried out. No papers with information on Trichinella spp. circulating in animals or humans have been published regarding the situation in Guyana, Surinam, French Guiana, Venezuela and Paraguay. Considering the growth of the guinea pig meat market in the Andean region, and the high prevalence of the disease reported in free range pigs and wild boars, as well as other game animal species, it is important to focus on the role of biosecurity and risk management, while improving meat market regulations, and detection of infection prior to consumption, in order to reduce the risk of transmission of this zoonotic disease to humans.

Conocimientos y prácticas asociadas a transmisión de triquinelosis, hanta y equinococosis quística / Knowledge and practices associated with transmission of trichinellosis, hanta and cystic equinococcosis

RESUMEN Objetivo: Asociar el nivel de conocimiento y prácticas (hábitos y costumbres) responsables de la transmisión de Triquinelosis, Síndrome Cardiopulmonar por Hantavirus y Equinococosis quística (Hidatidosis), en habitantes de los sectores rurales. Material y método: Estudio descriptivo correlacional de corte transversal en el que se aplicó una encuesta a 149 habitantes residentes en el área rural de las localidades de Curacautín, Lonquimay y Melipeuco, a través de muestreo no probabilístico por conveniencia, durante los meses de diciembre 2013 a enero 2014. Resultados: La población posee mejores conocimientos de Triquinelosis y Hanta que Hidatidosis, 64,9, 72,8 y 39,3%, respectivamente; los hábitos y costumbres para Hanta e Hidatidosis alcanzan mejores prácticas (84% cada una) que para Triquinelosis (69,5%); la población reconoce correctamente reservorio, fuente de infección y mecanismos de transmisión en Triquinelosis y Hanta, pero en menor grado Hidatidosis; respecto de los hábitos y costumbres, aun desconociendo los fundamentos teóricos, reportan buenas prácticas, aunque mantienen algunas que favorecen la transmisión de infecciones predominantemente en Triquinelosis. Conclusión: El nivel de conocimiento no asegura buenas prácticas, además los conocimientos y prácticas siguen un patrón empírico más que cognitivo, respaldados por aspectos culturales y del entorno. Esto sugiere mayor participación de los equipos interdisciplinarios en comunidades aisladas para fortalecer la promoción, educación y refuerzo de las buenas prácticas de acuerdo con sus condiciones sociales e incentivar la adherencia a conductas que reduzcan el riesgo de la transmisión de zoonosis.

ABSTRACT Objective: To associate the level of knowledge and practices (habits and customs) responsible for the transmission of trichinellosis, Hanta and cystic echinococcosis (hydatidosis), among rural population. Material and method: Cross-sectional correlational descriptive study in which 149 residents of the rural areas of Curacautín, Lonquimay and Melipeuco were surveyed, using non-probability convenience sampling from December 2013 to January 2014. Results: The population has better knowledge of trichinellosis and Hanta than hydatidosis, 64.9%, 72.8 and 39.3% respectively; they also have better practices regarding Hanta and hydatidosis (84% each) than regarding trichinellosis (69.5%). The population recognizes correctly the reservoir, source of infection and transmission mechanisms of trichinellosis and Hanta, and to a lesser degree hydatidosis; they report good practices regarding habits and customs, even ignoring theoretical foundations, but maintain some practices that favor the transmission of infections related to trichinellosis. Conclusion: The level of knowledge does not ensure good practices. Additionally, knowledge and practices follow an empirical rather than cognitive pattern, rooted on cultural and environmental aspects. This suggests greater participation of interdisciplinary teams in isolated communities to strengthen the promotion, education and reinforcement of good practices in accordance with their social conditions and to encourage adherence to behaviors that reduce the risk of the transmission of zoonoses.

[Knowledge and practices regarding cystic echinococcosis and trichinellosis in users and companions of users of the Hospital Comunitario de Salud Familiar El Carmen, Biobio Region, Chile]. / Conocimientos y prácticas sobre hidatidosis y triquinosis en usuarios y acompañantes del Hospital Comunitario de Salud Familiar El Carmen, Región del Biobío, Chile.

We surveyed users of the Hospital Comunitario de Salud Familiar El Carmen and their companions to analyze knowledge and practices regarding cystic echinococcosis and trichinellosis. Most people recognized risky practices. Previous attendance at talks and working in agricultural-livestock enhanced the knowledge. Age, sex and formal education were not associated with the knowledge. Knowledge was not associated with practices.

Conocimientos y prácticas sobre hidatidosis y triquinosis en usuarios y acompañantes del Hospital Comunitario de Salud Familiar El Carmen, Región del Biobío, Chile / Knowledge and practices regarding cystic echinococcosis and trichinellosis in users and companions of users of the Hospital Comunitario de Salud Familiar El Carmen, Biobio Region, Chile

We surveyed users of the Hospital Comunitario de Salud Familiar El Carmen and their companions to analyze knowledge and practices regarding cystic echinococcosis and trichinellosis. Most people recognized risky practices. Previous attendance at talks and working in agricultural-livestock enhanced the knowledge. Age, sex and formal education were not associated with the knowledge. Knowledge was not associated with practices.

Introducción: La comuna de El Carmen, Región del Biobío, presenta factores de riesgo para la hidatidosis y la triquinosis, como un alto índice de pobreza, actividad principalmente agropecuaria y carencia de plantas faenadoras de animales. Método: Se analizó, a través de una encuesta a los usuarios y acompañantes del hospital de El Carmen, el conocimiento y las prácticas relacionadas con esas enfermedades. Resultados: La mayoría de los encuestados reconoció conductas de riesgo. La asistencia previa a charlas y el trabajo en actividades agropecuarias se asoció a mayor conocimiento de la hidatidosis y triquinosis, respectivamente. No hubo asociación entre edad, género o nivel educacional con el conocimiento de las enfermedades. Tampoco hubo asociación entre el conocimiento y las prácticas. Conclusiones: Los resultados sugieren la necesidad de mejoras educacionales, así como también intervenciones públicas, especialmente en sectores más lejanos.

Trichinella spiralis infection and transplacental passage in human pregnancy.

Infection by Trichinella spp. during pregnancy still arises many questions. Worldwide an important number of trichinellosis outbreaks have occurred over time where pregnant women were or might have been involved. Parasitological and immunoserological parameters, clinical background and transplacental passage of Trichinella spiralis of six pregnant women, from different outbreaks, were studied. Serum samples were collected from patients and their children: at pregnancy, at delivery, newborn and/or infant (≥1 year-old). Total immunoglobulins (Igs) and isotypes against excretion-secretion products from muscle larvae (ESP-ML) and newborn larvae (NBL) of T. spiralis were detected by immunoserological techniques. Sera helminthocytotoxic activity against NBL was evaluated by antibodies dependent cell cytotoxicity assay. Patients presented similar clinical symptoms. Mother's sera showed differences in the presence and titres of Igs, IgE, IgG1 and IgG4 against NBL surface. All of them showed helminthocytotoxic activity against NBL, four in progesterone dependent manner and the other two by antibodies. Igs anti ESP-ML were present in mother's sera, with differences in the isotypes detected. Anti-ESP-ML IgE, IgA and/or IgM in newborn sera were observed. Three out of four infants showed specific Igs; one even at 8 years-old. The distinct immune response between mothers at pregnancy evidenced by sera helminthocytotoxic activity and immunoserological parameters may be associated with a different parasite burden exposition. Specific Igs observed in newborns would be a suggestion of transplacental passage of this Igs and/or their production by the fetus. Specific Igs found in infants sera suggests that newborn larvae transplacental passage is possible.

Parasitic zoonoses present some risks with low-temperature cooking of pork.

A recent article in this journal documents enhanced sensory qualities of pork cooked to low temperatures. The aim of this letter is to point out that the incidence of Trichinella spiralis in many countries and the more widespread incidence of Toxoplasma gondii present a concern for the adoption of low temperature cooking of pork unless extended cooking times are used.

[Assessment of transmammary transmission of Trichinella patagoniensis in BALB/c mice]. / Evaluación de la transmisión galactógena de Trichinella patagoniensis en ratones BALB/c.

Since 1916 to date, it has been suspected that vertical transmission of parasites from the genus Trichinella could occur in pregnant or lactating women during the parenteral phase of infection. The aim of the present study was to evaluate the transmammary transmission of T. patagoniensis in BALB/c mice. Twenty 7-week-old BALB/c mice were distributed into two groups of 10 individuals each, depending on the time of gestation when they were infected, 15 or 18 days after detection of the vaginal plug. Each group was subdivided into two subgroups of 5 mice each, which were given an oral dose of 100 or 500 infective larvae respectively. Euthanasia and subsequent artificial digestion was performed in the pups and the dams. No T. patagoniensis L1 larvae were found in any of the offsprings analyzed. The observed results suggest that vertical transmission of T. patagoniensis would not be possible in BALB/c mice.

Trichinella infection in wild boars (Sus scrofa) from a protected area of Argentina and its relationship with the presence of humans.

In Argentina, Trichinella infection has been documented in humans and animals of several provinces since 1930. This zoonotic parasite infection has been recently detected in humans and pigs of a region historically considered as Trichinella-free, suggesting the spread of these pathogens. The aim of the present work was to investigate the presence of Trichinella infection in wild boars (Sus scrofa) and in the human population living in a protected area. Trichinella infection has been investigated by serology (in humans and wild boars) and by artificial digestion of wild boar muscles. The isolated Trichinella larvae have been identified at the species level by multiplex PCR. A geographical information system has been used to collect environmental data. The results showed the circulation of Trichinella spiralis in wild boars with a low parasite burden, and suggest the influence of human behavior on the transmission. The transplacental passage of parasite is postulated. It follows that the declaration of region as Trichinella-free should be carefully established by means of extensive monitoring programs, not only in humans and domestic animals but also in wildlife.

Evaluation of the risk of transmission of Trichinella in pork production systems in Argentina.

Recently, there has been interest in programs that certify pork production practices that minimize the risk of exposure of pigs to Trichinella spiralis. Certification might be useful for reducing the risk of human trichinellosis from pork in Argentina, but more information is needed on pig production practices and sources of Trichinella infection in Argentinian pigs. In this study, 21 pig farms were assessed for Trichinella infection including some farms using total and partial confinement management, and others with pigs raised exclusively outdoors. A total of 3224 muscle samples were collected from pigs raised on these farms and tested to determine the presence of T. spiralis larvae by artificial digestion. Serum samples from the same 3224 pigs were tested for antibodies to T. spiralis by ELISA. For each farm, a questionnaire was completed summarizing information about management factors and this information was used to assess risk factors for exposure of T. spiralis. Based on the results, pigs raised outdoors were more likely to be infected than pigs raised in total or partial confinement (p< or =0.05). Pigs fed waste products containing meat were 12.5 times more likely to be infected than pigs not fed waste containing meat (p<0.01). The role played by rats in transmission of Trichinella is unclear; however, on farms with evidence of wild animals and access of pigs to wildlife carcasses, the prevalence of Trichinella infection was significantly higher. All pigs raised under good hygienic and sanitary conditions were negative for Trichinella infection by both artificial digestion and ELISA.

Trichinella spiralis / Trichinella spiralis

A Trichinella spiralis é um verme nematódeo intestinal, de que se conhecem várias espécies e que vive em estado larval nos músculos dos animais. É transmitido ao homem pela carne de porco. A triquinose é uma doença provocada no homem pela triquina. A triquinose manifesta-se nos casos graves, alguns dias apenas após a ingestão de carne contaminada. Traduz-se por catarro intestinal violento, semelhante à cólera ou à disenteria, dores de estômago, mal estar geral e vômitos biliosos ou mucosos. O quadro clínico modifica-se logo que os vermes penetram nos músculos: estes se tornam, então, muito sensíveis e dolorosos, dando sensação de rigidez e de fraqueza. A constatação de eosinofilia no sangue e o exame microscópico de uma biópsia podem ajudar no diagnóstico da doença. A cura geralmente verifica-se após o encistamento da triquina, que é lenta e necessita de vários meses de convalescença. Enquanto as triquinas permanecem no intestino, pode-se tentar expulsá-las por meio de laxativos fortes. Ainda não existe, porém, remédio específico para o estágio de encistamento intramuscular: o médico deverá limitar-se a manter as forças do paciente. Para afastar os perigos de triquinose, deve-se cozer a carne de porco cuidadosamente: acima de 70ºC, as triquinas são mortas, realmente. Esta temperatura, porém, deve alcançar, igualmente, o interior da carne: assim, é preciso cozer completamente a carne até que se tome cor branca ou cinza.

Trichinella spiralis / Trichinella spiralis

A Trichinella spiralis é um verme nematódeo intestinal, de que se conhecem várias espécies e que vive em estado larval nos músculos dos animais. É transmitido ao homem pela carne de porco. A triquinose é uma doença provocada no homem pela triquina. A triquinose manifesta-se nos casos graves, alguns dias apenas após a ingestão de carne contaminada. Traduz-se por catarro intestinal violento, semelhante à cólera ou à disenteria, dores de estômago, mal estar geral e vômitos biliosos ou mucosos. O quadro clínico modifica-se logo que os vermes penetram nos músculos: estes se tornam, então, muito sensíveis e dolorosos, dando sensação de rigidez e de fraqueza. A constatação de eosinofilia no sangue e o exame microscópico de uma biópsia podem ajudar no diagnóstico da doença. A cura geralmente verifica-se após o encistamento da triquina, que é lenta e necessita de vários meses de convalescença. Enquanto as triquinas permanecem no intestino, pode-se tentar expulsá-las por meio de laxativos fortes. Ainda não existe, porém, remédio específico para o estágio de encistamento intramuscular: o médico deverá limitar-se a manter as forças do paciente. Para afastar os perigos de triquinose, deve-se cozer a carne de porco cuidadosamente: acima de 70ºC, as triquinas são mortas, realmente. Esta temperatura, porém, deve alcançar, igualmente, o interior da carne: assim, é preciso cozer completamente a carne até que se tome cor branca ou cinza.(AU)

Trichinella spiralis is an intestinal nematode which many species are known and that lives in its larval stage in the animal' s muscles. It is transmitted to man by pork's meat. Triquinose is a disease caused in man by Trichinella spiralis appers in grave cases, some days afire the ingestion of the contaminated meat. The signs are violent intestinal catarrh, like a cholera or dysentery, stomachache, distress and mucous or bilious vomiting. The clinical picture modifies as soon as the worms enter the muscles: they become sensible and painful, giving the sensation of rigidity and weakness. The presence of eosinefilin in blood and the microscope exam of a biopsy can help in the diagnosis of the disease. The cure ofien can be seen after triquina encystment that is slow and take many months of convalescence. While the Trichinella spiralis remain in the intestine, we can try to expel them with strong laxatives. There are no specifics medicines to the intramuscular encystment stage yet. The doctor should just try to keep the patient' s strength. To keep out the dangers of the triquinose, we should cook pork's meat careflly: over 70° the Trichinella re- .ally die. This temperature however should reach equally the inside of the meat, so, it is necessary to cook the meat completely until it becomes white orgrey. (AU)

First record of human trichinosis in Chile associated with consumption of wild boar (Sus scrofa).

The first South American case of human trichinosis, resulting from the consumption of roast wild boar (Sus scrofa) is reported in Chile. The patient presented fever, diarrhea, myalgias, facial edema, sub-conjunctival reddening, photophobia, eosinophilia, and elevated glutamic oxalacetic transaminase. The diagnosis was confirmed by two immunoenzymatic tests (ELISA) using somatic and excretion-secretion antigens.

First record of human trichinosis in Chile associated with consumption of wild boar (Sus scrofa)

The first South American case of human trichinosis, resulting from the consumption of roast wild boar (Sus scrofa) is reported in Chile. The patient presented fever, diarrhea, myalgias, facial edema, sub-conjunctival reddening, photophobia, eosinophilia, and elevated glutamic oxalacetic transaminase. The diagnosis was confirmed by two immunoenzymatic tests (ELISA) using somatic and excretion-secretion antigens.

Triquinosis: importancia del caso índice / Trichinosis: importance of the index case

Se reporta el caso de un paciente varón de 28 años, previamente sano, proveniente de Curicó, que tras haber ingerido carne de cerdo faenado en domicilio, inició un cuadro clínico y exámenes de laboratorio altamente sugerentes triquinosis. Se notificó de inmediato a la sección de Epidemiología del ministerio de Salud, con lo que se logró precozmente encontrar al resto de los afectados y decomisar la carne contaminada para evitar nuevos casos, destacando de esta forma la importancia del caso índice. Posteriormente se apoyó el diagnóstico con test de ELISA para trichinella spiralis, y se inició el tratamiento anti parasitario con albendazol y sintomático, con lo cual el paciente evolucionó satisfactoriamente. Se destaca la importancia del diagnóstico precoz y de la oportuna notificación a las autoridades sanitarias cuando se tiene un cuadro clínico compatible, y exámenes de laboratorio generales, como hemograma y los niveles de CPK alterados, aun cuando no se disponga de exámenes serológicos específicos, como ELISA para triquinosis

[Trichinellosis approaching the year 2000]. / La trichinellosi verso il 2000.

Relevant information is given on the Ninth International Conference on Trichinellosis held in Mexico City from 19 to 22 August 1996. The Conference was organized in 4 Symposia and 8 Workshops. One-hundred-thirty scientific contributions (on the epidemiology, biology, systematics, immunology, biochemistry, molecular biology, pathology, legislation and control) were presented by 90 research scientists coming from 21 countries.

Actualidades sobre la triquinelosis equina / Current facts about horse trichinellosis

La triquinelosis es una zoonosis causada por parásitos del género Trichinella que es trasmitida principalmente por la ingestión de carne de animales como el cerdo, oso y zorro entre otros. Sin embargo, en Europa se han reportado varios brotes debido al comsumo de carne de caballo. La presencia del parásito no ha sido demostrada de manera directa en la carne de estos animales, sin embargo, la identificación de las especies de Trichinella (T. spiralis T. britovi y T. nativa) involucradas en estos brotes ha sido posible a partir de biopsias tomadas de individuos que consumieron carne de caballo. Recientemente, se identificaron por primera vez larvas de T. spiralis en caballos sacrificados en un rastro del Estado de México, presentando así evidencia directa de la infección de estos animales con el parásito. Por otro lado empleando extractos totales o antígenos TSL-1 de T. spiralis se han detectado anticuerpos en contra de Trichinella en caballos sacrificados en rastros de diferentes paises de Europa así como en México. Asimismo, la infección con varias especies de Trichinella se ha logrado reproducir experimentalmente en caballos y los resultados obtenidos son importantes en el desarrollo de métodos de diagnóstico que permitan estimas la prevalencia de esta infección en caballos cuya carne se destina para el consumo tanto animal como humano y eventualmente instrumentar medias para el control de la trasmisión de la triquinelosis por carne de caballos

Clinical aspects of infection with Trichinella spp.

Isolated cases and outbreaks of infection with Trichinella spp. occur frequently throughout the world, sometimes resulting in fatalities. The clinical presentations of signs and symptoms are remarkably constant for most of the species of Trichinella, but in infections with Trichinella nativa and Trichinella britovi, classical symptoms of trichinellosis may be absent. It is important to be able to correlate the clinical presentation of trichinellosis with the life cycle of these helminths in order to make an accurate diagnosis. Knowledge of the epidemiology of the disease enables the physician to identify other potential cases, since most epidemics can be traced back to a common source of raw or undercooked meat. A comprehensive summary relating the most important clinical variables is presented graphically for easy reference to the text. Symptoms and signs are considered in relation to severity of infection. Laboratory findings and diagnostic techniques, including new modalities (e.g., DNA and antigen detection), are discussed. A discussion of treatment and preventive measures concludes our review.

[Trichinosis in children. A report of 8 cases]. / Triquinosis en el niño. Informe de 8 casos.

A comparative study between 8 cases of trichinosis and 16 of typhoid fever is presented in this paper; all of them were attended in the INP during the period from 1970 to 1990. The diagnosis was established under epidemiologic, clinic and laboratory bases. The range of age of these patients was from 10 to 14 years, from low socioeconomic status; half of the cases had history of in take of infested pork meat. The most characteristic symptoms were: fever, myalgia and limitation of muscular movements. Leukocytosis in addition to eosinophilia were found in 7 cases. Correlation with indirect hemagglutination, CIEF positive test for Trichinella spiralis and biopsy of muscle was done. Thiabendozole therapy was utilized for treatment with satisfactory evolution, except in one case that developed progressive and fatal neurologic damage.

[The epidemiological profile of trichinosis in the state of Zacatecas]. / Perfil epidemiológico de la triquinosis en el estado de Zacatecas.

A compilation of data from the cases which have occurred in Zacatecas during the period between 1976 and 1987 has been made. It describes the endemia and locates the problem in time, person and space. Because of their impact, magnitude and consequences the more important data of the outbreaks is presented. The first case of trichinosis diagnosed in Zacatecas was in 1975; since then there have been other outbreaks; four in 1978, with the highest mortality of 33 per cent which occurred in Laguna del Carretero a community of Villanueva. During the period between 1979-1988 there have been 17 more, among these a death occurred in Valparaiso and, in Pozo de Gamboa there was an abortion in a 20-year-old female patient. This infection has higher frequency in urban areas. The most affected group age is 15-44 years; in this group there have been 51 percent of the cases occurring in females in a proportion of 1:1.8. Seven of the 36 countries in the state of Zacatecas, such as Zacatecas, Villanueva, Valparaiso, Panuco, Jerez, Jalpa and Guadalupe, have reported cases; the countries are listed by frequency. The transmission in most of the cases was caused by ingestion of raw pork products, in a fen undercooked pork meat. The diagnosis was confirmed in sixty cases by the conservation of the parasite in muscle biopsy and oronyosistis which represent a 38 percent of the reported total cases. Six cases was confirmed by histologic sections and immunoelectrophoresis test.