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.

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.

[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.

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

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.

[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.

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

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.

[Determination of anti-HTLV-I/II antibodies: Experience in 28,897 blood donations in Buenos Aires]. / Determinación de anticuerpos anti HTLV-I/II: experiencia en 28,897 donantes de sangre en Buenos Aires.

PURPOSE: The first human retrovirus, HTLV-I, was isolated in 1980; HTLV-II was described later. The former is endemic in southwestern Japan, the Caribbean and equatorial Africa; whereas the latter prevails in intravenous drug addicts, being also endemic in American indian populations. Both viruses are either sexually transmitted, from mother to child mainly by breast-feeding, by blood transfusion or by sharing contaminated needles. With regard to transmission, since they are intracellular viruses, it occurs only when whole blood or cellular components are transfused; this is not the case when either plasma or plasma derivatives are used. The likelihood of transmission decreases as the storage time increases. HTLV-I is associated, at least, with two diseases: adult T-cell leukaemia/lymphoma (ATLL), and the tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM). ATLL occurs after a latency period of 20 to 30 years; whereas the incubation period ranges from 3 to 5 years in the case of the neurological disease. Most individuals infected with the virus remain healthy; the risk of developing the hematological complication is 2-4% whereas it is below 1% in the case of TSP. No clear association of HTLV-II with any known disease has been reported as yet. In this study, we have assessed the prevalence of HTLV-I and HTLV-II in the sera of the blood donors who have come to our Division, with the aim of avoiding the spreading of this oncogenic virus by transfusion. This study could serve as a measure of the infection in the general population. MATERIAL AND METHODS: A total of 28,897 samples were analyzed from May 1993 to January 1995. Anti-HTLV-I/II antibodies were analyzed by the method of passive agglutination of gelatin participles (PA). Samples which reacted were tested again by the same method, and those reacting for the second time were further confirmed by Western blot (WBT), a method with the ability to differentiate between antibodies anti-HTLV-I and anti-HTLV-II. RESULTS: Of the 28,897 samples, 47 were repeatedly reactive by PA (0.16%). Analysis by WBT resulted in 10 reactive results with HTLV-I (0.035%), 2 reactive results with HTLV-II (0.007%); in one sample it could not be determined whether the anti-HTLV-I or anti-HTLV-II antibody was present. Of the remaining samples, 21 were non-reacting, whereas 13 were indeterminated. CONCLUSION: Prevalence of HTLV-I and HTLV-II seropositive blood donors is low and similar to that found in other non-endemic countries. We believe that routine evaluation of anti-HTLV-I and HTLV-II antibodies in blood donors would be warranted in our country, since transmission of the viruses by transfusion of blood components has been clearly shown. It is possible that the recipients of the reactive units do not develop the disease. Nevertheless, these individuals constitute an important source of virus dissemination, both during the perinatal period and by sexual intercourse. In fact, advise to seropositive donors would prevent transmission by these routes. Lastly, it should be noticed that investigation of anti-HTLV-I/II antibodies could result in a surrogate method for detecting other viral infections transmitted by these routes.

Cyclophosphamide effect on Paracoccidioidomycosis in the rat

La paracoccidioidomicosis es una enfermedad endemica fungica ampliamente distribuida en Latino-America. La ciclofosfamida ha sido usada como potente imunosupresor para modular la respuesta inmune, en um modelo experimental infectado con Paracocidioides brasiliensis. Ratas macho buffalo/sim endocriadas de 250-300 gr. de peso, fueron inoculadas por via intracardiaca con 5.10 elevado a sexta potencia celalas de P. brasiliensis en fase levaduriforme...

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.

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.

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.

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.

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.

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.

Immunoperoxidase tracing of Junin virus neural route after footpad inoculation.

To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10-15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

[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.