Abstract. The persistence of Trypanosoma cruzi tissue forms was detected in the myocardium of seropositive individuals clinically diagnosed as chronic chagasic patients following endomyocardial biopsies (EMBs) processed by immunohistochemical (peroxidase-anti-peroxidase [PAP] staining) and molecular (polymerase chain reaction [PCR]) techniques. An indirect immunofluorescent technique revealed antigenic deposits in the cardiac tissue in 24 (88.9%) of 27 patients. Persistent T. cruzi amastigotes were detected by PAP staining in the myocardium of 22 (84.6%) of 26 patients. This finding was confirmed with a PCR assay specific for T. cruzi in 21 (91.3%) of 23 biopsy specimens from the same patients. Statistical analysis revealed substantial agreement between PCR and PAP techniques (k ϭ 0.68) and the PCR and any serologic test (k ϭ 0.77). The histopathologic study of EMB specimens from these patients revealed necrosis, inflammatory infiltrates, and fibrosis, and made it possible to detect heart abnormalities not detected by electrocardiogram and/or cineventriculogram. These indications of myocarditis were supported by the detection of T. cruzi amastigotes by the PAP technique or its genome by PCR. They suggest that although the number of parasites is low in patients with chronic Chagas' disease, their potential for heart damage may be comparable with those present during the acute phase. The urgent necessity for testing new drugs with long-term effects on T. cruzi is discussed in the context of the present results.

Abstract.A clinical, parasitologic, and serologic study carried out between 1988 and 1996 on 59 acute-phase patients in areas of western Venezuela where Chagas' disease is endemic showed 19 symptomatic patterns or groups of symptoms appearing in combination with different frequencies. The symptomatic pattern with the highest frequency was that showing simultaneously fever, myalgia, headache, and Romaña's sign, which was detected in 20% of the acute-phase patients. Asymptomatic individuals and patients with fever as the only sign of the disease made up 15% and 11.9% of the total acute cases, respectively. Statistical correlation analysis revealed that xenodiagnosis and hemoculture were the most reliable and concordant of the five parasitologic methods used; these two methods also showed the highest proportions in detecting any clinical symptomatic pattern in acute-phase patients. A similar high reliability and concordance was obtained with a direct agglutination test, an indirect immunofluorescent antibody test, and an ELISA as serologic tests, which also showed a higher proportion of positive detection of clinical patterns than parasitologic methods (P Ͻ 0.001). It is recommended that individuals coming from endemic areas showing mild and/or severe clinical manifestations should be suspected of being in contact or having been in contact with Trypanosoma cruzi, be referred for parasitologic and serologic evaluations to confirm the presumptive clinical diagnosis of acute Chagas' disease, and start specific treatment. The epidemiologic implications of the present findings are discussed and the use of similar methodology to evaluate other areas where Chagas' disease is endemic is suggested.Shortly after describing Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, Carlos Chagas created the basis for future study of the disease that was named after him. He was the first to recognize the role that triatomine bugs play in the transmission of the infection and provided information about its pathology, symptomatology, epidemiology, and geographic distribution.

We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at ؉93 downstream of the 3 end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the ؉93 site quantitatively terminate at a fail-safe terminator located further downstream at ؉250. Inactivation of Rnt1p (an RNase III involved in processing the 3 end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the ؉93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies.Understanding the mechanism of transcription termination requires reproduction of the termination process in vitro where appropriately detailed experiments are feasible. For study of RNA polymerase I (PolI) termination in the yeast Saccharomyces cerevisiae, we initially used a whole-cell extract to catalogue sites of RNA 3Ј end formation downstream of the 3Ј end of mature 25S rRNA (15). Beginning at the 3Ј end of 25S rRNA (position Ϫ1), we mapped additional termini to ϩ93, to about ϩ250, and to a further site in the vicinity of ϩ350. Eventually we settled on the ϩ93 site for further study since it possessed several attributes expected of the primary terminator for PolI transcription. For example, the ϩ93 site behaved kinetically as a terminator rather than as a processing site. Addition of actinomycin D to a whole-cell transcription reaction abruptly stopped both transcription and accumulation of termini at ϩ93, as expected of a true terminator. In contrast, accumulation of the 3Ј end of 25S rRNA continued after transcription stopped, as expected of a processing site (15). In addition, later work using beaded templates demonstrated that transcripts are released at ϩ93 rather than just being paused on the template (14, 16).We have also found that both the structure and the mechanism of action of the ϩ93 terminator are similar to those of PolI terminators that have been studied in vertebrates, indicating conservation during evolution. Similarities include the fact that in both yeast and vertebrates, termination occurs just upstream of a protein binding site (Reb1p in yeast and TTFI in mice or humans). These terminator proteins function only when bound in one orientation, and they all utilize paired Myb homology domains as DNA binding elements. In addition to the protein binding site, it is now recognized that PolI terminators contain a second DNA element which codes for the last 10 to 12 nucleotides (nt) of the terminated transcript and which is often T rich. Studies on mechanism, in both yeasts and vertebrates, show that termination at these sites occurs in two steps, pause and release; the protein binding site acting primarily as the pause element, while the upstream region controlling re...

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We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.