Real-time PCR strategy for rapid discrimination among main lymnaeid species from Argentina.

Snails of the Family Lymnaeidae act as an intermediate hosts of Fasciola hepatica worldwide. The taxonomy of lymnaeid species is relevant for epidemiological studies and molecular strategies are increasingly used for that purpose. This work presents the first report of a real-time PCR approach used to identify the most important lymnaeid species in the Southern Cone of South America. Species discrimination is based on the sequence polymorphism located within the helix E10-1 of the variable region V2 of the 18S rRNA genes, which yields amplicons with clearly different melting temperatures. This procedure minimises the risk of carry-over contamination because it does not require post-PCR manipulations, and the whole protocol can be completed in less than 4h with a single snail foot as starting material. This method was successfully carried out in a blind study that included a panel of 20 Galba truncatula, 5 Lymnaea viatrix, 5 Lymnaea diaphana and 5 Pseudosuccinea columella specimens from different endemic areas for fasciolosis. This molecular approach constitutes a key laboratory tool complementing ecological studies that ultimately will promote more efficient control strategies.

Comparison of two coprological methods for the veterinary diagnosis of fasciolosis

Compararam-se a sensibilidade dos métodos padrão de sedimentação fecal (MPSF) e modificado da tamisação e coloração das fezes (MTCF)-quatro tamises, ambos empregados no diagnóstico de infecção por Fasciola hepatica. Amostras de fezes foram obtidas de 51 bovinos oriundos de uma área endêmica por fasciolose no Sudoeste da Argentina. Cada amostra foi colocada em um recipiente contendo formalina 5%. Oito mililitros da suspensão, equivalente a 2g de fezes, foram usados em cada método testado, registrando-se o número de ovos por amostra. A porcentagem de amostras positivas pelo MTCF (27/51) foi maior que a apresentada pelo MPSF (11/51), o que representou 60% de amostras falso-negativas. A porcentagem de concordância pelos dois métodos foi 41%. A complexidade do MPSF pode diminuir sua sensibilidade pela perda de ovos durante o processamento. Estes resultados confirmaram que o MPSF subestima a contagem de ovos e que o método MTCF é mais confiável.

Comparison of two coprological methods for the veterinary diagnosis of fasciolosis / Comparação de dois métodos coprológicos para diagnóstico da fasciolose

The sensitivity and utility of a standard faecal sedimentation method (FSM) and a modified stool sieving staining method (FSSM), both currently employed for the diagnosis of Fasciola hepatica infection were compared. Faecal samples were obtained from 51 bovines of an endemic area for fasciolosis in Southwestern Argentina. Each sample was placed in a recipient containing 5% formalin. Eight millilitres of the suspension, equivalent to 2g of faeces, were used for each of the two methods tested. The number of eggs found per sample was recorded. The proportion of positive samples obtained by the FSSM (27/51) was significantly higher than that by the FSM (11/51) (P<0.05). The percent of agreement between methods was 41%. Over a total of 27 positive samples detected by the FSSM, the FSM missed 16, yielding 60% false negative samples. The FSSM enhanced 2.5 times the sensitivity of diagnosis. The complexity of the FSM may decrease its sensitivity through missing and loss of eggs during sample processing. These results confirmed that the commonly used FSM underestimates the prevalence and the egg output in cattle and that the FSSM is a more reliable diagnostic method.(AU)

Compararam-se a sensibilidade dos métodos padrão de sedimentação fecal (MPSF) e modificado da tamisação e coloração das fezes (MTCF)-quatro tamises, ambos empregados no diagnóstico de infecção por Fasciola hepatica. Amostras de fezes foram obtidas de 51 bovinos oriundos de uma área endêmica por fasciolose no Sudoeste da Argentina. Cada amostra foi colocada em um recipiente contendo formalina 5%. Oito mililitros da suspensão, equivalente a 2g de fezes, foram usados em cada método testado, registrando-se o número de ovos por amostra. A porcentagem de amostras positivas pelo MTCF (27/51) foi maior que a apresentada pelo MPSF (11/51), o que representou 60% de amostras falso-negativas. A porcentagem de concordância pelos dois métodos foi 41%. A complexidade do MPSF pode diminuir sua sensibilidade pela perda de ovos durante o processamento. Estes resultados confirmaram que o MPSF subestima a contagem de ovos e que o método MTCF é mais confiável.(AU)

Estudio del foco en un caso de fasciolosis humana en Neuquén / A focus study from a case of human fascioliasis in Neuquén

El objetivo del trabajo fue realizar un estudio de foco relacionado con un caso de fasciolosis humana ocurrido en abril de 2002 en Loncopué, Neuquén, Argentina. La confirmación diagnóstica se efectuó en mayo de 2002 por el test de ELISA. En noviembre de 2002 se realizó un muestreo en el área rural donde se ubicaba la vivienda de la paciente, ya restablecida, y se le tomó una nueva muestra de sangre. El suero de la paciente continuó reactivo para antígenos de Fasciola hepatica. Se muestrearon plantas de berro para detectar metacercarias. Se recolectaron caracoles en cuatro canales de riego conectados a un canal principal. Los caracoles fueron trasladados vivos para su identificación, medición y examen de infección. Se recolectaron 35 muestras fecales de ganado de cría. No se observaron metacercarias en las hojas de berro examinadas (n=222). Se recolectaron 130 caracoles identificados como Lymnaea viatrix y 2 de 101 ejemplares (2%) estaban infectados con larvas de F. hepatica. Las prevalencias en el ganado adulto fueron: 100% (10/10) para caprinos, 82% (9/11) para ovinos y 86% (6/7) para bovinos. El número de huevos eliminados por las cabras (mediana = 20.7; Q1=6.2; Q3=34.5) y ovejas (4, 18, 13) infectadas, resultó mayor que el eliminado por vacas (0.3; 0.3; 1.7) (p<0.01). La práctica de control local no tuvo efecto aparente en este caso, por lo que deberían revisarse los calendarios de tratamiento y los antiparasitarios utilizados. Los resultados muestran que el ganado criado a pequeña escala por los pobladores debe incluirse en los programas de control. Se discute la posible importancia de la fasciolosis humana en Argentina. (AU)

Estudio del foco en un caso de fasciolosis humana en Neuquén / A focus study from a case of human fascioliasis in Neuquén

El objetivo del trabajo fue realizar un estudio de foco relacionado con un caso de fasciolosis humana ocurrido en abril de 2002 en Loncopué, Neuquén, Argentina. La confirmación diagnóstica se efectuó en mayo de 2002 por el test de ELISA. En noviembre de 2002 se realizó un muestreo en el área rural donde se ubicaba la vivienda de la paciente, ya restablecida, y se le tomó una nueva muestra de sangre. El suero de la paciente continuó reactivo para antígenos de Fasciola hepatica. Se muestrearon plantas de berro para detectar metacercarias. Se recolectaron caracoles en cuatro canales de riego conectados a un canal principal. Los caracoles fueron trasladados vivos para su identificación, medición y examen de infección. Se recolectaron 35 muestras fecales de ganado de cría. No se observaron metacercarias en las hojas de berro examinadas (n=222). Se recolectaron 130 caracoles identificados como Lymnaea viatrix y 2 de 101 ejemplares (2%) estaban infectados con larvas de F. hepatica. Las prevalencias en el ganado adulto fueron: 100% (10/10) para caprinos, 82% (9/11) para ovinos y 86% (6/7) para bovinos. El número de huevos eliminados por las cabras (mediana = 20.7; Q1=6.2; Q3=34.5) y ovejas (4, 18, 13) infectadas, resultó mayor que el eliminado por vacas (0.3; 0.3; 1.7) (p<0.01). La práctica de control local no tuvo efecto aparente en este caso, por lo que deberían revisarse los calendarios de tratamiento y los antiparasitarios utilizados. Los resultados muestran que el ganado criado a pequeña escala por los pobladores debe incluirse en los programas de control. Se discute la posible importancia de la fasciolosis humana en Argentina.

A frequent TG deletion near the polyadenylation signal of the human HEXB gene: occurrence of an irregular DNA structure and conserved nucleotide sequence motif in the 3' untranslated region.

While screening for new mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase, a TG deletion (deltaTG) was found in the 3' untranslated region (3'UTR) of the gene, 7 bp upstream from the polyadenylation signal. Examination of DNA samples of 145 unrelated Argentinean individuals from different racial backgrounds showed that the deltaTG allele was present with a frequency of approximately 0.1, compared with the wild-type (WT) allele. The deletion was not associated with infantile or variant forms of Sandhoff disease when present in combination with a deleterious allele. Total Hex and Hex B enzymatic activities measured in individuals heterozygous for deltaTG and a null allele, IVS-2 + 1G-->A (G-->A), were approximately 30% lower than the activities of G-->A/WT individuals. Analysis of the HEXB mRNA from leukocytes of deltaTG/WT individuals by RT-PCR of the 3'UTR showed that the deltaTG allele is present at lower level than the WT allele. By polyacrylamide gel electrophoresis, it was determined that a PCR fragment containing the +TG version of the 3'UTR of the HEXB gene had an irregular structure. On inspection of genes containing a TG dinucleotide upstream from the polyadenylation signal we found that this dinucleotide was part of a conserved sequence (TGTTTT) immersed in a A/T-rich region. This sequence arrangement was present in more than 40% analyzed eukaryotic mRNAs, including in the human, mouse and cat HEXB genes. The significance of the TG deletion in reference to Sandhoff disease as well as the possible functional role of the consensus sequence and the DNA structure of the 3'UTR are considered.

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection.

The level of beta-hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2 + 1 G-->A, and a 4-bp deletion, delta CTTT782-785. These mutations, and a 16-kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the delta CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.