Trans-sialidase inhibition assay for the detection of Trypanosoma cruzi infection in blood donor samples from Argentina.

BACKGROUND AND OBJECTIVES: Conventional serology tests for Trypanosoma cruzi blood banks screening are neither sensitive nor specific enough, and currently no gold standard assay is available. Trans-sialidase inhibition assay (TIA) detects neutralizing antibodies against T. cruzi trans-sialidase. Conventional serology inconclusive, positive and negative blood donor samples were evaluated by employing TIA as a supplementary test. MATERIALS AND METHODS: Three hundred and twenty-one blood donor samples were tested using a combination of assays. Based on the results of testing, these were divided into a number of groups. All samples were tested by TIA. RESULTS: In conventional serology inconclusive samples 48.1% were TIA-positive, 1/54 conventional serology positive samples was TIA-negative. All negative samples from donors without epidemiological risks were TIA-negative; 1/48 was positive in those with epidemiological risk. CONCLUSION: Trans-sialidase inhibition assay application in blood banks may be useful to resolve inconclusive samples, and thus improves donor counseling and allows individual re-entry. The use of TIA in samples from negative conventional test donors but positive epidemiological antecedents may contribute to decrease transfusional risk.

Brain inflammatory exudate in Junin virus-infected rats: its characterization by the immunoperoxidase (PAP) technique.

Morphologic changes in cyclophosphamide (CY)-suppressed vs. control non-suppressed new-born rats infected i.c. with XJC13 strain of Junin virus were compared and the cells involved in CNS lesions were identified by the PAP technique. Fifty per cent of the control rats exhibited widespread cerebral necrosis vs. only 15% of the immunosuppressed animals. The first cells to reach Junin virus-infected CNS in controls were T lymphocytes, which destroyed viral antigen-laden target neurons and astrocytes. B lymphocytes and macrophages, presumably attracted by viral antigen and/or by lymphokines, made their appearance a day or two later. Activated macrophages phagocytosed necrotic cells and perhaps exerted a cytotoxic effect upon target neural cells, whereas the actual role of B lymphocytes requires further explanation. In CY-treated rats, cerebral lesions were smaller and the cellular exudate, though similar, proved much scantier than in controls. A similar extent of cerebellar necrosis was observed in both groups.

Antibodies to Trypanosoma cruzi among blood donors in Buenos Aires, Argentina.

OBJECTIVES: The aim of this work was to study the prevalence of anti-Trypanosoma cruzi in the blood donor population in Buenos Aires, to compare the relative sensitivity and specificity of the two screening tests used and to confirm the results with a third assay. MATERIAL AND METHODS: Between May 1995 and July 1999, 64,887 blood donor consecutive samples were screened with the following commercial tests: indirect hemagglutination (IHA) (Polychaco, Buenos Aires, Argentina) and enzyme-linked immunosorbent assay (ELISA) (40,222 with Chagatek, Organon Teknika, Buenos Aires, Argentina, and 24,665 with Chagas EIA, Abbott, São Paulo, Brazil). Repeatedly reactive samples in one or both tests were analyzed with a third method: dot blot (Bio Chagas, Gador, Buenos Aires, Argentina) or particle agglutination (Serodia, Fujirebio, Tokyo, Japan). Sera that reacted in at least two tests were considered positive. RESULTS: The seroprevalence was 2.66% (1744 samples were reactive for one or both screening tests), and 1.46% (949 samples) were confirmed positive. The ELISAs proved to be more sensitive (relative sensitivity: 99.67-99.71%) whereas 192 samples (0.47%) were IHA false-negatives (relative sensitivity: 79.77%). Relative specificity for EIA was 98.47--99.23% and for IHA 99.85%. CONCLUSIONS: Results suggest the need of performing two screening tests for Chagas disease in blood banks from endemic areas and the importance of a third confirmatory assay to avoid unnecessary medical counseling.

Cyclophosphamide effect on paracoccidioidomycosis in the rat.

Paracoccidioidomycosis is an endemic fungal disease widely distributed throughout Latin America. The potent immunosuppressor cyclophosphamide (CY) has been used to modulate host immune response to Paracoccidioides brasiliensis in an experimental model. Inbred male Buffalo/Sim rats weighing 250-300 g were inoculated with 5 x 10(6) P. brasiliensis cells of the yeast phase form by intracardiac route. One group of animals was treated with 20 mg/kg body weight at days +4, +5, +6, +7, +11 and +12 post-infection (pi.), while a control group was infected alone. No mortality was recorded in either group. Treated rats presented: a) a decrease in granuloma size, which contained less fungal cells; b) a lack of specific antibodies up to 35 days pi., and c) a significant increase in the footpad swelling test (DTH) against paracoccidioidin. Splenic cell transfer from CY-treated P. brasiliensis-infected donors to recipients infected alone led to a significant increase in DTH response in the latter versus untreated infected controls. Likewise, in treated infected recipients transferred with untreated infected donor spleen cells, footpad swelling proved greater than in controls. Thus, it would seem that each successive suppressor T lymphocyte subset belonging to the respective cascade may be sensitive to repeated CY doses administered up to 12 days pi.. Alternatively, such CY schedule may induce the appearance of a T cell population capable of amplifying DTH response.

Experimental coccidioidomycosis in the immunosuppressed rat.

C. immitis inoculated rats are known to develop infection restricted to lung whereas cyclophosphamide (CY) treatment leads to widespread dissemination with considerable mortality. In this study, an attempt was made to elucidate the mechanisms involved in such behaviour. With this aim, spleen cells were transferred from infected CY-treated to infected untreated rats, achieving significant specific inhibition in footpad swelling to coccidioidin in recipients, attributable to a suppressor T cell subpopulation induced by greater fungal antigen concentration arising from widespread C. immitis dissemination in immunosuppressed animals. NK activity proved similar regardless of CY treatment. Lastly, chronically infected rats presented increased colony forming units count after several weekly doses of CY, as happens in immunosuppressed patients harbouring a previous infection.

Cyclophosphamide effect on coccidioidomycosis in the rat.

Cocidioidomycosis is a systemic mycosis, endemic in arid areas of the American continent. The rat was employed as an experimental host, since it had been shown to reproduce human lesions and present a chronic course of disease with granulomas mainly restricted to lungs. Given the influence of immunosuppressive therapy on the clinical course of human coccidioidomycosis, we studied the effect of cyclophosphamide (CY) in the experimental rat model. Accordingly, animals were inoculated with 400 Coccidioides immitis arthroconidia of the Acosta strain, by intracardiacal route. As single CY doses failed to alter the course of disease, three schedules were used: A) 4 daily doses of 20 mg/kg each, prior to C. immitis inoculation; B) 4 similar daily doses after infection; and C); 6 doses of 20 mg/kg each, given from day +1 to +4 then on days +8 and +9, post infection (pi), taking day 0 as the time of fungal inoculation. The first two schedules inhibited antibody formation up to day 28 pi, without modifying cellular response to coccidioidin as measured by foodpad swelling. Initially, there was greater fungal spread than in controls receiving C. immitis alone, which proved self-limiting in the latter. In contrast, schedule C led to 55% mortality with both humoral and cellular response abrogation, accompanied by extensive C. immitis dissemination. Histology disclosed significant alterations, such as the persistence of primary infection sporangia, corresponding to the acute stage of coccidioidomycosis in the absence of granuloma development. Therefore, the observed depression in cellular immunity seems responsible for the lack of inflammatory reaction capable of restricting sporangia proliferation in tissues which, in turn, enhances pathogen spread and mortality rate.

Action of antithymocyte serum on Junin virus infection in rats.

Two-day-old rats resistant to intracerebral (i.c.) infection with XJ strain of Junin virus (JV), were rendered sensitive to JV by treatment with antithymocyte serum (ATS). The mortality reached 80%, the virus titres in brain were higher and the serum neutralizing antibodies dropped but brain lesions were absent throughout. The same host was susceptible to XJCl3 strain infection, which induced lethal encephalitis manifested by severe necrotic foci in cerebellum. ATS treatment conferred significant protection against this strain; the mortality was 63%, viral titres in brain remained unchanged but the lesions were mild as compared with non-treated animals. It seems likely that the XJ strain allowed the 2-day-old rat to develop serum antibodies against JV, while the XJCl3 strain unleashed an immunopathologic response.

Development of specific immune response in mice infected with Junin virus.

Different parameters of specific immune response involved in the resistance to intracerebral Junin virus (JV) infection were studied in adult BALB/c mice. The relationship of virus replication to production of antiviral antibodies, to occurrence of cytotoxic T cells and to development of delayed-type hypersensitivity response was evaluated. Spleen cytotoxic T cells were assayed by 51Cr-release method on virus-infected H2 compatible targets. Effector T cells were detected on day 2, reached peak concentrations by day 6 and declined on day 10. These cells seemed responsible, at least in part, for virus clearance from the infected target organ, since virus could not be recovered from the brain in any sample taken on days 2, 5, 6, 8, 10, 15 and 20 post infection (p.i.). All three main antibody classes common in viral infection were present. Serum antibodies appeared later than the T cell cytotoxic response. Neutralizing antibodies and those detected by immunofluorescence prevailed in the IgG fraction, whereas the IgM antibody class was reactive in complement fixation assay. Challenge of infected mice with JV did not result in production of delayed-type hypersensitivity as measured by footpad swelling irrespective of the route of sensitization. The possible interpretations of these findings are discussed in connection with the resistance of adult mice to JV infection.

[Is TT virus (TTV) a true hepatitis virus cause?]. / Virus TT (TTV). Es un verdadero causante de hepatitis?

TT virus (TTV) was first detected in the blood of three patients with elevated serum alanine aminotransferase following transfusion who were negative for all known hepatitis viruses. This virus exhibited hepatotropism, and its titers correlated with elevation in serum aminotransferase concentration suggesting that it was a true hepatitis virus. Moreover, it was demonstrated that the presence of TTV DNA is not associated with biochemical or histologic evidence of liver injury. The virus has been found worldwide with a high prevalence in the general population and there is evidence that it may be transmitted by parenteral exposure to blood, enterally and transmitted from mother to child. An association between TTV infection and acute or chronic hepatitis or other diseases has not been consistently observed.

Neural spread of Junin virus in intraperitoneally inoculated rats.

On the basis of an already demonstrated Junín virus (JV) neural route after peripheral footpad infection of newborn rats, here we attempted to determine the viral pathway following intraperitoneal inoculation. As from the 2nd week post-infection, neurological disease developed reaching 84% mortality at 30 days. Immunoperoxidase labeling of viral antigen, concomitantly with infectivity assays and histological examination, was carried out in serially harvested samples. Whenever infectivity was detected, whether by viral rescue from coculture or by conventional isolation, viral antigen staining was achieved. Infective JV was present at threshold levels in spleen and liver from days 2 to 10, and in blood from days 5 to 15. In neural tissues, viral antigen was initially disclosed at day 5 in thoracic rachideal ganglia and related spinal cord segments. From day 7 thereafter, the entire spinal cord was involved; at this stage, first evidence of viral infection was found in brain stem, with subsequent spread to other encephalon structures at day 10. According to harvested samples, no significant differences were found in labeled cell percentages at thoracic vs cervical or lumbar levels of spinal cord. In contrasts, greater involvement of cerebral cortex versus brain stem, hippocampus or cerebellum was demonstrated shortly before death. Although JV antigen was overwhelmingly predominant in neurons, no morphological changes were apparent in such cells. Since rachideal spinal ganglia and spinal cord infection invariably preceded viral spread to encephalon, concomitantly with viral clearance from lymphoreticular organs and blood, a neural pathway seems warranted.

[Prevalence of Trypanosoma cruzi antibodies in blood donors]. / Prevalencia de anticuerpos anti-Trypanosoma cruzi en donantes de sangre.

Blood transfusion is the second most common transmission route of Chagas' disease in endemic areas. Discrepancies between the available diagnostic kits are common, which indicates that a single test is not satisfactory. The aim of this work was to study the seroprevalence of anti-Trypanosoma cruzi markers, to compare sensitivity and specificity of the two screening assays currently in use and to confirm the results with a third test. A total of 20,860 volunteer blood donors were studied. Donations were screened with indirect hemagglutination assay (IHA) and enzyme immunoassay (EIA). Repeatedly reactive samples were assayed with an EIA carried out on strips, to which a mixture of T. cruzi antigens was applied as an horizontal line (DB). Sera that reacted in at least two tests were considered positive. A total of 576 samples were reactive for one or both screening tests (2.76%) and 391 of them (1.87%) were confirmed positive. EIA proved to be more sensitive, with no false negative results (100% sensibility), whereas 98 samples (0.47%) were IHA false negative (74.93% sensibility). Specificity for EIA was 99.3% and for IHA 99.8%. In our case, almost 0.9% of donated blood is discarded because of false reactive anti-T. cruzi results; two thirds correspond to false positive EIA and one third to false positive IHA. It is important to note that in our population we have not registered false negative results for EIA but there were false negative IHA. This fact implies that although the first method is less specific, it is much more sensitive. It is important to confirm the screening results in order to avoid unnecessary donor counselling and permit future donations. The DB test employed in our study results a useful alternative for this purpose.

Evaluation of HTLV-I/II infection in blood donors in Buenos Aires.

HTLV-I and HTLV-II are two related retroviruses that are transmitted by sexual contact, breast feeding, blood transfusion and needle sharing. In this study the prevalence of HTLV-I and HTLV-II was evaluated in voluntary blood donors as a measure of the infection in the general population. Samples were tested by a gelatine particle agglutination test and repeatedly reactive samples were confirmed by Western blot tests (WBT), enriched with recombinant rgp21, rgp461 y rgp4611 proteins, which differentiates HTLV-I and HTLV-II antibodies. Of 19,426 samples, 40 were repeatedly reactive by particle agglutination (0.21%). When analyzed by WBT, 6 met the criteria for HTLV-I (0.036%), 2 for HTLV-II (0.01%) and 1 for HTLV-I/II, 13 samples were indeterminate and 18 were negative. The prevalence is low and comparable to that from non endemic countries. Screening for anti HTLV-I/II antibodies is necessary to prevent transmission through blood transfusions.

[Differentiation among strains of Junín virus by intraperitoneal infection in the rat]. / Diferenciación de cepas de virus Junín por la infección intraperitoneal en la rata.

The 2-day-old rat is known to resist intracerebral infection with the XJ prototype strain of Junin virus, but 95-100% mortality results when infected with the attenuated XJC13 strain. When this animal was inoculated by intraperitoneal route, behaviour was diametrically opposite: the XJ strain proved lethal, while de XJC13 led to low mortality. Studies on mortality, virus titer in different organs, and anti-viral humoral response in 2-day-old rats infected with Junin virus strains were carried out in order to use this system as a new attenuation marker. Mortality rates recorded for rats inoculated with either strain, were markedly different, being 84% in the XJ-infected group and barely reaching 17% in the XJC13 group. Brain viral titers were higher in the former group than the latter (10(5.26) PFU/ml vs. 10(3) PFU/ml at day 17 pi). For this reason, viral replication may be used as a virulence marker in this experimental model. Antibody levels were also higher in the XJ group most likely due to greater viral replication. The above findings support the use of the 2-day-old rat as a biologic attenuation marker since susceptibility to infection is strain-dependent.

[Infection in rats by the intraperitoneal route with Junín virus]. / Infección en ratas por vía intraperitoneal con virus Junín.

The susceptibility of the rat, infected at various ages by intraperitoneal route with the XJ prototype strain of Junin virus was studied two day-old animals showed maximum mortality, being the most suitable dose 10(3) LD50. Antiviral humoral response at 35 days pi was tested in survivors of all ages. Highest neutralizing antibody titers were found in those infected up to 4 days of age. This host behaves quite differently when infected by intracerebral route with the same XJ strain.

[Immunopathology induced in the rat by Junin virus]. / Inmunopatología inducida en la rata por virus Junín.

Intra-cerebral infection of the 10-day-old rat with the XJ prototype strain of Junin virus induces an immunopathological encephalitis with 100% mortality. In contrast with previous observations, our present work with antithymocyte serum (ATS) demonstrates a pathological role for the cellular immune response in this experimental model. As regards ATS treatment, 3 schedules were employed, the most efficient being daily 0.01 ml/g weight doses from day -1 to day +9, then +12, +14 and +16, taking day 0 as the time of virus infection. Survival reached 54% and the average day of death was delayed 12 days (Table 1). No differences were recorded in brain viral titres in treated vs untreated infected controls (Table 2). Lastly, splenocyte transfer from infected 10-day-old rats, to infected 2-day-old animals, which are known to develop persistence without death, led to 40% mortality in recipients vs 0% in 2-day-old non-transferred infected controls. Therefore, it may be concluded that: a) encephalitis in the 10-day-old rat is immunological in nature and b) transfer of lymphocytes to infected 2-day-old rats, induces disease and death.

[Protection against encephalitis in rats caused by a pathogenic strain of the Junin virus, using peripheral inoculation of an attenuated strain]. / Protección contra la encefalitis producida en ratas por una cepa patógena de virus Junín por inoculación periférica con una cepa atenuada.

Argentine Hemorrhagic Fever manifests itself in man either subclinically or in hemorrhagic or neurological forms, mortality reaching 20%. Although Candid 1 strain is undergoing pilot trials, current therapy still resorts to convalescent serum administration. A neurological model was used to evaluate protection conferred by the attenuated XJC13 Junin virus strain. Newborn rats inoculated intraperitoneally (ip) prove resistant, whereas 8-12 day-old animals infected by intracerebral route with the XJ prototype strain suffer 100% mortality with neurological signs. The aim of this study was to achieve protection in this model and attempt to elucidate the mechanisms involved in resistance. It was observed that the longer the inoculation challenge interval, the greater was the survival percentage. In protected animals, brain viral titres were 3 log lower than in challenged controls, while XJC13 infected unchallenged controls presented low CNS values throughout. Neutralizing antibody levels were not significantly different in experimental versus challenged control groups, ruling out any secondary booster effect on protected rats. Neither the transfer of immunoserum nor of endogenous or exogenous interferon altered mortality. However, when splenocytes from rats infected 10 days previously were transferred prior to XJ challenge, survival was increased to 50%, but there was no gain in protection when cells were treated with antithymocyte serum plus complement. Consequently, protection in this neurological model can be attributed to a cellular immune response.

[Effect of cyclophosphamide on experimental infection of rats with 2 strains of the Junin virus]. / Acción de la ciclofosfamida en la infección experimental de la rata con dos cepas de virus Junín.

The course of viral infection in rats of several ages after intracerebral inoculation with two strains of Junin virus, as well as the effect of an immunosuppressor was studied. The survival rate in 2-day-old rats was 95%, which fell to 45% in cyclophosphamide-treated infected animals (Figure 1a). However, barely 5% of these rats inoculated with the XJCl3 strain survived, while the cyclophosphamide suppressive treatment increased the rate to 36% (Figure 1b). This contrasting behaviour suggested that the XJ strain produces a subclinical infection in 2-day-old rats and the host immune mechanisms are responsible for recovery from the viral infection. Adequate immunosuppression converts sublethal experimental infections into lethal infection, accompanied by persistent viral replication in the target organ (brain) and suppression of anti-viral antibodies (Figure 2a). On the other hand, the inoculation of 2-day-old rats with the XJCl3 Junin virus may give rise to an immunopathology avoidable by CY treatment. In 10-day-old rats both strains of Junin virus caused a direct pathology, not modified by CY treatment (Table 3). This treatment failed to change susceptibility or virus concentration in the brain, but specific antibodies were considerably reduced. In the case of 26-day-old rats, there was total resistance to viral infection which remained unchanged after CY treatment.

[Role of macrophages in the dissemination of the Junin virus in the rat]. / Papel del macrófago en la diseminación del virus Junín en la rata.

The 2-day-old rat is known to be susceptible to infection by ip route with the XJ strain of Junin virus but resists inoculation with XJC13 strain (85% vs 15% mortality). In order to determine whether peritoneal macrophages play a role in modulating the course of infection, viral replication in adherent peritoneal cells infected with either strain was studied. XJ was found to replicate 30-fold as regards XJC13 at day 3 pi. Besides, silica blockage of peritoneal macrophages was also evaluated. Following silica treatment, 60% survival was recorded for XJ-infected animals vs 15% for untreated infected controls. No significant differences were recorded for silica-treated XJC13-infected rats vs untreated infected controls. These preliminary findings indicate that, upon replication within the host's macrophages at the peritoneal inoculation site, virus spreads readily to reach the target organs. Effective silica blockage lends support to this pathway, as shown by significantly greater survival in XJ-infected rats.

Attenuation parameters for Junin virus in the newborn rat.

To characterize a virus strain as attenuated, both biologic and biochemical criteria are necessary. In the case of Junin virus, the 2-day-old rat has proved to be a biologic attenuation marker as regards mortality. Here we studied the behaviour of the prototype XJ vs the attenuated XJC13 strain inoculated by either ic or ip route to determine differential hematologic and splenic parameters. Humoral immune response against SRBC was also investigated. By either route XJ caused significant leucocytosis, while the other hematologic parameters remained unchanged. No alterations were found following XJC13 infection. XJ produced significant splenomegaly whereas XJC13 had no effect. Similarly PFC anti-SRBC count was decreased during XJ infection but not after XJC13 infection. These differences between the pathogenic XJ and the attenuated XJC13 strain may be attributed to the former's greater spread. The drop in PFC could be due to spleen dysfunction and/or viral effects on the cell subpopulation involved.