Fundamento: El diagnostico precoz de la Hidatidosis Humana mediante metodos inmunologicoa ha sido una de las actividades mas importantes desplegadas por los Programas de Lucha contra la Hidatidosis en las iireas endemicas de la Argentina para mejorar el pronostico del paciente hidatidico. Es asi de gran interes definir con exactitud las ventajas y limitaciones de las tecnicas de Elisa y DD5 en el diagnostico de la hidatidosis en poblaciones sin sintomas clinicos de la enfermedad. Metodos: Para ello se evaluan la sensibilidad, especificidad y valor predictivo de ambos metodos. estudiandose serologicamente 499 pobladores de una comunidad rural de la Provincia de Rio Negro. Se estudian por imagenes utilizandose ecografia. radioscopia y tomografia computada los pobladores con serologia reactiva EIE y/o DD5 y tambien un grupo control seleccionado en base a su serologia no reactiva. Se deriva a cirugia los casos con Imagen compatible con Hidatidosis. Resultados: La tecnica de DD5 se demuestm como 100 % especifica pero con una sensibilidad de solo el 31 %. EIE, por su parte. a un nivel de corte de 8 DE presenta una especificidad del 97 % con una sensibilidad del 63 %. Conclusiones: Se destaca el valor de Ia ecografia como primer diagnostico por imagenes, en tanto la tornografia en la presente experiencia, no arrojo mayor numero de imagenes positivas permitiendo solo confirmar los hallazgos ecograficos.

At present, Trichinella spiralis is the only species of this genus reported from South America. Herein, we detail a molecular analysis of a new encapsulated isolate of muscle larvae of Trichinella, found in a mountain lion (Puma concolor) coming from the Patagonia, Argentina. We studied three DNA regions previously probed to be useful for the identification of all eleven recognized Trichinella genotypes: expansion segment 5 (ES5), cytochrome c-oxidase subunit I (COI) and 5S ribosomal DNA intergenic spacer region (5S ISR). BLAST searches with these DNA sequences showed that the mitochondrial and nuclear ribosomal regions most closely resemble other Trichinella sequences available in GenBank. However, they did not exactly match any of the eleven recognized genotypes. The phylogenetic analysis from COI and 5S ISR sequences showed that the mountain lion isolate is grouped with encapsulated members, in concordance with morphological data. Furthermore, this new isolate was located at the base of the encapsulated genotypes, signifying that it is an old genotype that could have emerged earliest in this group. These data strongly suggest that this isolate from the Patagonia represents the twelfth genotype (T12) described in the genus Trichinella. Nevertheless, further studies are necessary to adequately establish this isolate as a unique genotype.

In Argentina, a country considered non-endemic for hepatitis E virus (HEV) infection, serologic evidence of HEV infection has been observed in different human population groups. In other countries, a high degree of genetic relatedness has been observed between human and swine HEV genotype 3 sequences, suggesting zoonosis as one probable route of infection. This is the first identification of swine HEV in South America. HEV RNA was detected and sequenced in the ORF 1 and ORF 2 regions from swine fecal samples from a herd located in Pergamino, in the province of Buenos Aires. These strains all group into genotype 3 and exhibit a close relationship to two novel HEV variants previously identified in Argentina from sporadic acute cases of non-A to -C hepatitis in humans. In addition, using a modified commercial ELISA, the presence of anti-HEV antibodies was surveyed in five provinces across the country and all five showed a prevalence of HEV antibodies, ranging from 4% to 58%. The results suggest that swine could be an important reservoir for virus transmission in Argentina as has been suggested for other nonendemic areas. The Argentine human strains and swine strain described in this article seem to be closely related to a human Austrian strain, suggesting a potential European origin of HEV infection in these cases.

In 2000 Guarnera et al. proposed using ELISA in canine faeces collected from the ground to detect dogs infected with Echinococcus granulosus, thus determining sheep farms with active transmission. The objective was to evaluate the prevalence of E. granulosus infection in sheep farms of the Patagonia. Sheep farms were randomly selected in the Provinces of Río Negro, Chubut, Neuquén, Santa Cruz and Tierra del Fuego (areas with control programs) and La Pampa (comparison area). From one to three samples of fecal matter were obtained for each sheep farm, which were processed by means of copro-ELISA test with confirmation of positive samples by copro-Western blot. A total of 1042 samples were obtained from 352 sheep farms, 26 (7.3%) proving positive. Of these 5 (6.3%) were from La Pampa, 9 (13.8%) from Neuquén, 4 (4.7%) from Río Negro, 2 (2.9%) from Chubut, 1 (5.9%) from Santa Cruz and 5 (13.9%) from Tierra del Fuego. The identification of parasitized dogs is an essential activity upon which rests the strategy of control and surveillance. Arecoline tests or coproantigen test with fecal matter obtained directly from the dog contribute information on individual prevalence, while the use of coproantigens detected in ground-collected samples transfers the dog unit of observation to units of greater epidemiological value. In the present experience, the technique employed seems promising for its application in systems of epidemiological surveillance of cystic echinococcosis and in drawing a baseline on which to measure the progress of control programs in the Argentine Patagonia in subsequent years.

In 2000, two cases of human trichinellosis were detected in the Sierra Grande area of Rio Negro province, Argentina. As part of an investigation of the aetiology of these cases, 300 pigs slaughtered for consumption in the area between 2000 and 2002 were checked for Trichinella infection, by artificial digestion of a muscle sample. Twelve (5.6%) four (7.3%) of the 55 checked in 2000, five (4.8%) of the 105 investigated in 2001, and three (2.1%) of the 140 investigated in 2002 - were found infected. Blood samples were collected from other pigs aged > 6 months old, so that sera could be tested, in ELISA and by western blotting, for anti-Trichinella antibodies. Of the 181 animals checked in the initial serological survey, 36 (19.9%) were found seropositive for Trichinella. When 35 of the seronegative pigs were re-checked 6 months later, three (8.6%) were found to have seroconverted.Four (15.4%) of 26 local rodents, caught in Sherman-type traps, were also found positive when checked for infection by artificial digestion. It appears that about 20% of pigs in the study area are infected each year, this high level of transmission being sustained by a high prevalence of infection in the local rodent populations.

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30–66, 114–150, 181–217 and 247–283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

Random Amplified Polymorphic DNAs, (RAPDs) are used to study the occurrence of Trichinella britovi and T5 among domestic animals in the Province of Buenos Aires, Argentina and to assess the genetic diversity among isolates of T. spiralisfrom this area in a number of infected hosts. All the local isolates proved to be T. spiralis. Six of the eight primers used indicate that the Buenos Aires isolates are distinct from each other as they produce a considerable number of polymorphic bands. Our overall estimates are relatively higher than other intraspecific distances previously estimated within species of this genus and among T. spiralis isolates. Such high degrees of variability observed among local isolates and between isolates from Buenos Aires and Spain should be taken into account when defining isolates within this species, and considering differences in the epidemiology of T. spiralis.

A novel tandemly repeated DNA structure of Toxoplasma gondii that meets the requirements assigned for satellital DNA was characterized. A DNA fragment of 1002 bp contains two different elements of repetitive DNA families named ABGTg7 and ABGTg8.2. Both repeats are members of a more complex tandem structure where ABGTg7-like monomers can be arranged either as direct tandems or flanked by other related or non-related repeats. Pulse-field gel electrophoresis analysis showed that these repeats hybridize with the largest T. gondii chromosomes. Bal31 sensitivity assays indicated that these elements are located near the telomeres and along other regions too. Five genomic lambda phages were isolated and two different completed clusters of the repeated structure were analyzed.

ABSTRACTTheToxoplasma gondiirhoptry protein Rop2 was expressed inEscherichia colias a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2196–561). Humoral response duringToxoplasmainfection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196–561as the antigen substrate. The analyzed sera were divided according toT. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG+IgA−IgM−;n= 35), group B (IgG+IgA+IgM+;n= 21), group C (IgG+IgA+IgM−;n= 5), and group D (IgG+IgA−IgM+;n= 16). Twenty-sixT. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2196–561was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. SinceT. gondiiRop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2196–561would be suitable for use in diagnostic systems, in combination with otherT. gondiiantigens, to detect specific IgG, IgA, and IgM antibodies.

We report the potential use of a specific Toxoplasma gondii DNA probe (ABGTg7). We applied a dot blot hybridization assay to blood samples for the diagnosis of cerebral toxoplasmosis (CT), acute toxoplasmic lymphadenopathy (ATL), and disseminated toxoplasmosis in transplant recipients (TRs). We studied a total of 84 individuals: 38 patients and 46 controls. We found positive hybridization signals for 12 (66.7%) of 18 patients with confirmed CT, 9 (52.9%) of 17 patients with ATL, and 2 (66.7%) of 3 TRs. PCR assays were performed in parallel for patients with ATL, resulting in T. gondii DNA detection for 10 patients (58.8%). A comparative study between dot blot and PCR assays performed with the blood of mice that had been experimentally infected with tachyzoites gave similar results: 60 and 70% positive results, respectively. Finally, the sum of positive values obtained by both DNA tests (dot blot assay plus PCR) increased the rate of positivity for ATL patients to 76.4%. These results demonstrate that the T. gondii ABGTg7 repetitive DNA element is an additional useful resource for diagnosing Toxoplasma parasitemia in patients with CT and ATL and in TRs. Thus, our ABGTg7-based dot blot test may lead to an improvement in T. gondii detection methods in patients with acute toxoplasmosis.

An outbreak of Brucella melitensis in a family was studied. From the fourteen family members who ate unpasteurized goat cheese nine became ill. Patients included four females and five males of 8 to 75 years old. In seven of the patients the diagnosis was confirmed by positive blood culture for B. melitensis biovar 1. All the patients were analyzed by standard tube agglutination (STA) and standard tube agglutination with 2-mercaptoethanol (STA-2ME) tests at the time of diagnosis. In six of the patients, ELISA assays were used to assess the humoral immune anti-protein and anti-lipopolysaccharide (LPS) responses. Anti-LPS IgG antibodies were detected in all of the patients. Anti-proteins IgG antibodies were present at significant levels in all the studied patients including the STA-2ME negative ones.

BACKGROUND:The early diagnosis of Human Hydatidosis through immunologic methods has been one of the most important activities displayed by the Programme of Struggle (fight) in endemic areas of Argentina, to improve the prediction of the Hydatid patient. It is of great interest to define exactly the advantages and limitations of the DD5 and Elisa techniques in the diagnosis of hydatidosis in inhabited areas with no clinic symptoms of the disease.METHODS:For this, the sensibility, the specification and predictive value of both methods has been evaluated, studying serologically 499 inhabitants from a rural community in the Province of Río Negro. The inhabitants with reactive serology to EIE and/or DD5 are studied by means of images using US, RX and TC. A controlled group is selected according to its un-reactive serology. The cases compatible with Hydatidosis are directed to surgery.RESULTS:The DD5 technique is shown as 100% specific but with a sensibility of only 31%; on the other hand, EIE at a cutting level of 8 DE presents a specification of 97% with a sensibility of 63%.CONCLUSIONS:It is important to underline the value of the US as the first image prognosis, while the TC, in the present experience, did not show a great number of positive images, allowing only to confirm the US findings.

An outbreak of Brucella melitensis in a family was studied. From the fourteen family members who ate unpasteurized goat cheese nine became ill. Patients included four females and five males of 8 to 75 years old. In seven of the patients the diagnosis was confirmed by positive blood culture for B. melitensis biovar 1. All the patients were analyzed by standard tube agglutination (STA) and standard tube agglutination with 2-mercaptoethanol (STA-2ME) tests at the time of diagnosis. In six of the patients, ELISA assays were used to assess the humoral immune anti-protein and anti-lipopolysaccharide (LPS) responses. Anti-LPS IgG antibodies were detected in all of the patients. Anti-proteins IgG antibodies were present at significant levels in all the studied patients including the STA-2ME negative ones.

Hydatidosis is a serious Public Health problem in the Province of Río Negro, Argentina. This situation has promoted the implementation of a control programme based on the canine systematic deparasitation with Praziquantel, health education, work control and care of persons. The accumulated experience of 15 years works, carried out to develop the last work--line mentioned above, is presented in this study. The activities includes the registry of human cases, the early identification of asymptomatic carries, the infected persons derivation to the hospital system in order to allow their appropriate treatment, the longitudinal follow up of the operated cases and the epidemiologic surveillance of the human hydatidosis situation. A network of laboratories was organized, as a support to satisfy the hospital demand, under the coordination of a central laboratory of reference and serologic surveys, among the groups at risk, were also carried out. The information registered indicates that a total of 60,078 serologic tests were carried out in the whole Province: out of them, 22,899 corresponded to hospital demand (DD5) and 37,179 to serologic surveys (originally latex, then DD5, and now Elisa). 65% of the cases, diagnosed by official register, is derived from rural areas to complexity hospitals, to be put under surgical treatment, and, out of these operated persons, only 3.9% is derived from the appearance of clinical symptomatology. A sustained reduction in the average days of stay, in lethality and in the rate of serologic prevalence in school children from 7 to 13 years of age, are achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

The basic strategy for development of hydatid struggle programs is in the actuality: Primary Attention of Health. In the present work and in this instance, it's arm a precocious detection system of hydatid disease, fixed in immunologic diagnostic by means of ELISA technical beginning with blood capillary samples, taken in filter paper by teachers and sanitary agents from official services of Rio Negro Province. 177 teachers and 45 sanitary agents were trained, correspondent to 25 schools, 3 lodging schools and 9 Hospitals all of them from rural area. 890 blood samples during the training were obtained. Lastly, the trained personal armed the system and they obtained 728 samples in the beginning of the Program. It hadn't statistical differences in the reactivity of both samples. The serological prevalence found was 1.32%. The activity displayed by teachers and sanitary agents permitted to detect 21 new cases it was the 20% of new cases diagnosed in this area in the period of work. The viability and the importance of the incorporation of non traditional effectors into the Hydatid Control Programs is discussed.

In South America programs to control hydatidosis caused by Echinococcus granulosus include the active search for asymptomatic patients through population surveys for the detection of antibodies against arc 5 antigens using the double diffusion arc 5 test (DD5). Though simple to perform and highly specific, DD5 is not practical for population studies due to the time lapse between testing and receiving results. This work evaluates the application of an enzyme immunoassay to screen sera for subsequent processing using DD5. The efficiency of an enzyme immunoassay screening/DD5 confirmation scheme vs. DD5 alone was compared within the framework of a control program. A total of 5,839 sera from residents of endemic areas was processed and 47 hydatid patients were detected by both schemes. The proposed enzyme immunoassay identified all sera having antibody activity against arc 5 antigens detectable by DD5 and ruled out 95.3% of sera which tested by DD5 would have produced negative results.

Programmes for the control of hydatidosis caused by Echinococcus granulosus in Argentina use an enzyme immunoassay (EIA) as a screening test for population surveys aimed at detecting asymptomatic patients. Persons thus selected are referred to health centres for the arc 5 double diffusion test and imaging techniques. One of the most costly procedures of these surveys is the collection of blood samples under field conditions; the possibility of collecting dry blood samples on filter paper was therefore investigated. In a survey of 497 rural inhabitants of an endemic area, the same number of hydatidosis cases (22) were identified by EIA using (i) serum samples and (ii) capillary blood samples obtained by finger prick and collected on filter paper. The latter system was both simpler and cheaper.