Maxicircle genomic organization and editing of an ATPase subunit 6 RNA in Trypanosoma cruzi.

The DNA sequence of a 5736-nucleotide (nt) Trypanosoma cruzi maxicircle fragment was determined. Sequence comparisons indicate that its 5' terminus is the homologue of the downstream portion of the NADH dehydrogenase subunit 7 gene and that its 3' region is homologous to the maxicircle unidentified reading frame II gene. The region between these two gene segments contains six additional genes that encode mitochondrial proteins, including ATPase subunit 6 (A6). Comparison of the A6 maxicircle DNA sequence with that of an A6 cDNA indicates that the A6 RNA is extensively edited throughout its length. A 49-nt sequence that could serve as template for transcription of a guide RNA for editing a segment of the A6 RNA was found in one of 24 minicircle variable regions sequenced. Moreover, the presence of an RNA having this sequence was demonstrated in an RNAse protection assay. This is the first identification of a guide RNA template in a T. cruzi minicircle. Taken together, our findings suggest that T. cruzi and Trypanosoma brucei brucei are phylogenetically closer to each other than they are to Leishmania tarentolae, despite the relative similarity of the life cycles of the latter and T. cruzi.

Regulatory sequences and a novel gene in the msp (GP63) gene cluster of Leishmania chagasi.

The surface protease GP63 of Leishmania chagasi is encoded by a cluster of more than 18 tandem major surface protease (msp) genes belonging to three classes (mspL, mspS, mspC). mspL and mspS transcripts are differentially expressed during parasite growth. RNAs from mspS genes predominate during stationary phase, the time when parasite virulence and GP63 expression are maximal. We hypothesized that the unique regions downstream of mspS genes contain signals important for gene expression. The 2.8 kb region between tandem mspS genes was found to contain an 882 bp open reading frame designated mag. Copies of mag were found downstream of all mspS genes in the cluster. mag hybridized faintly to bands on Northern blots and a fully processed mag cDNA was identified in a promastigote cDNA library, providing evidence that mag genes are expressed at low levels. Similar to mspS RNAs, the abundance of mag RNAs was greater in stationary phase than logarithmic phase organisms, although mag RNAs were less abundant than mspS RNAs throughout growth. Northern blots and enzyme assays of promastigotes containing plasmid constructs in which the beta-galactosidase gene was followed by sequences between mspS coding regions, either with or without mag and its downstream sequences, suggest these regions have several regulatory effects accounting for the growth-associated changes in mspS expression.

Postmortem diagnosis of autochthonous acute chagasic myocarditis by polymerase chain reaction amplification of a species-specific DNA sequence of Trypanosoma cruzi.

We report a fatal case of vector-transmitted acute Chagas' myocarditis in a seven-month-old child in south Texas. This diagnosis was not suspected during the three days of hospitalization that preceded the child's death, which was caused by heart failure. A diagnosis of acute myocarditis, probably of viral origin, was listed as the cause of death after cardiac tissue was examined microscopically at autopsy. One year after the death of the patient, a diagnosis of Trypanosoma cruzi myocarditis, based solely on morphological grounds, was made after newly prepared slides of cardiac tissue were examined. Seven years later, we confirmed the diagnosis of T. cruzi infection by using the polymerase chain reaction to amplify a species-specific genomic repetitive DNA sequence of the parasite from fixed cardiac tissue.

Use of the polymerase chain reaction for detecting Trypanosoma cruzi in triatomine vectors.

Determination of the rate of Trypanosoma cruzi infection in its triatomine vectors is an element in control programmes directed at reducing transmission of the organism to humans. Traditionally, T. cruzi has been detected in these insects by microscopical examination of intestinal contents or excreta. The sensitivity of this laborious process has not been defined because of the lack of a bench-mark method against which microscopical examination could be compared. The purpose of this study was to compare the sensitivity of a polymerase chain reaction (PCR) assay with that of microscopical examination for detecting T. cruzi in Triatoma infestans nymphs that had fed on patients with chronic Chagas disease. To this end, we analysed 54 pairs of samples, each containing 2 groups of 10 insects, obtained by feedings on 19 patients with chronic T. cruzi infection, 17 of whom were fed upon 3 times. One group of insects in each pair was analysed by PCR and the other by microscopical examination of excreta. Overall, the PCR assay gave positive results in 32 of 54 groups of insects examined (59%), whereas only 7 of 54 groups (13%) were positive by microscopical examination (P = 0.038). These results demonstrate that the PCR assay is significantly more sensitive for the detection of T. cruzi in triatomine vectors than is microscopical examination, and suggest that the PCR assay could be a useful tool in epizootiological studies.

Long-term outcome study of growth factor-treated pressure ulcers.

BACKGROUND: Exogenous application of growth factors have been reported in an attempt to accelerate healing of chronic wounds. Most of the trials were of brief duration with short to no follow-up periods. Long-term outcome studies are sparse for pressure ulcer therapies with success rates around 30% for both operative and nonoperative treatments. METHODS: Follow-up evaluations were performed serially up to 12 months for patients completing a 35 day blinded, placebo-controlled cytokine clinical trial of pressure ulcers. RESULTS: Fifty-four of 61 patients completed the follow-up period with 68.5% of the patients (37 of 54) being healed after 1 year. Of patients healing > or =85% during the active treatment phase, 84.6% were healed after 1 year compared with 61% of those that healed <85% during treatment (P <0.05). CONCLUSION: Long-term outcome was better in this growth factor trial than with surgical or standard nonoperative treatment of pressure ulcers. Since only patients receiving exogenously applied cytokines achieved >85% closure during the treatment phase of the trial, the excellent long-term outcome appears attributable to the cytokine therapy.

Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi.

The diagnosis of acute infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is generally made by detecting parasites by microscopic examination of fresh blood. Although highly specific, this approach often lacks sensitivity. Several years ago, PCR assays for the detection of T. cruzi were described, but the sensitivities and specificities of these tests have not yet been defined precisely. In the present study, we first compared the sensitivities of PCR methods that differ in sample processing as well as in the target sequences that are amplified. Then, we challenged eight mice with T. cruzi, and on 31 days over a 380-day period, we compared the ability of the PCR method with the highest sensitivity to detect parasites in blood with that of microscopic examination. During the acute phase of the infections, parasites were detected on average 3.9 days earlier by the PCR method than by microscopy. Furthermore, the infected mice were consistently positive by the PCR method during the chronic phase, while parasites were intermittently detected by microscopic examination during that period. Overall, among the 248 comparisons, in 84 the PCR method was positive and no parasites were seen by microscopic examination, whereas the reverse was true in only 1 case, a difference that is highly significant. These findings suggest that this approach should be in patients suspected of having acute Chagas' disease. Moreover, the higher sensitivity of the PCR method observed in both the acute and chronic phases of the T. cruzi infections in the mice that we studied indicates that this approach should be useful in evaluating experimental drugs in T. cruzi-infected laboratory animals.

Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.

The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.