gp72, the 72 kilodalton glycoprotein, is the membrane acceptor site for C3 on Trypanosoma cruzi epimastigotes.

We examined the interaction of complement component C3 with surface molecules on Trypanosoma cruzi. Five- to six-fold more C3 was bound to epimastigotes (Epi) than to metacyclic trypomastigotes (CMT) of strain M88. Epi and CMT were surface iodinated, then incubated in C8-deficient serum, and detergent lysates were applied to anti-C3 antibody that had been coupled to Sepharose. We found that 9.20-10.24% of applied 125I-Epi protein bound to anti-C3-sepharose, compared to 2.64% binding of 125I-CMT protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that C3 was attached to 125I-Epi protein by a covalent bond. Samples eluted from anti-C3-sepharose with hydroxylamine revealed a single, major, 72 kD band, suggesting that C3b attaches almost exclusively to the 72 kD glycoprotein of Epi by a hydroxylamine-susceptible ester bond. An antiserum was prepared from lysates of serum-treated Epi that had been affinity-purified on anti-C3-sepharose. This antiserum immunoprecipitated a single 72 kD component (gp72) from surface-iodinated Epi, and specifically recognized only gp72 from Epi in immunoblots. In contrast to the results with Epi, gp72 on CMT was not found to be an efficient acceptor molecule for C3 deposition. The results are the first to evaluate the acceptor site for C3 deposition on a parasite, and they show that gp72 on Epi, but not gp72 on CMT, serves as the preferential acceptor for C3 during antibody-independent alternative complement pathway activation.

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.

Evidence of Trypanosoma cruzi infection (Chagas' disease) among patients undergoing cardiac surgery.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas' heart disease, is transmitted by triatomine insects and by blood transfusion. The emigration of several million people from T cruzi-endemic countries to the United States has raised concerns regarding a possible increase in cases of Chagas' heart disease here, as well as an increased risk of transfusion-transmitted T cruzi. To investigate these 2 possible outcomes, we tested a repository of blood specimens from multiply transfused cardiac surgery patients for antibodies to T cruzi. METHODS AND RESULTS: Postoperative blood specimens from 11 430 cardiac surgery patients were tested by enzyme immunoassay, and if repeat-reactive, were confirmed by radioimmunoprecipitation. Six postoperative specimens (0.05%) were confirmed positive. Corresponding preoperative specimens, available for 4 of these patients, were also positive. The other 2 patients had undergone heart transplantations. Tissue samples from their excised hearts were tested for T cruzi by polymerase chain reaction and were positive. Despite the fact that several of these 6 patients had histories and clinical findings suggestive of Chagas' disease, none of them were diagnosed with or tested for it. Patient demographics showed that 5 of 6 positive patients were Hispanic, and overall, 2. 7% of Hispanic patients in the repository were positive. CONCLUSIONS: No evidence for transfusion-transmitted T cruzi was found. All 6 seropositive patients apparently were infected with T cruzi before surgery; however, a diagnosis of Chagas' disease was not known or even considered in any of these patients. Indeed, Chagas' disease may be an underdiagnosed cause of cardiac disease in the United States, particularly among patients born in countries in which T cruzi is endemic.

American trypanosomiasis (Chagas' disease) in Central American immigrants.

A survey was conducted to determine the prevalence of infection with Trypanosoma cruzi, the protozoan etiologic agent of American trypanosomiasis (Chagas' disease), among Nicaraguan and Salvadoran immigrants living in the Washington, D.C., area. The serum samples of study subjects were tested for reactivity with T. cruzi antigens in an enzyme-linked immunosorbent assay, and also tested for antibody specific for the 72 and 90 kilodalton (kDa) surface glycoproteins of the parasite in an immunoprecipitation and electrophoresis procedure. Xenodiagnosis using reduviid bugs to detect parasites, and clinical evaluations for cardiac and gastrointestinal disease were performed in patients in whom results of both serologic tests were positive. Of 205 subjects studied, 4.9 percent were infected with T. cruzi, and parasites were isolated from 50 percent of those in whom xenodiagnosis was attempted. No significant cardiac or gastrointestinal abnormalities were detected in the six infected patients who were evaluated clinically. These findings suggest that a sizable proportion of persons in this immigrant group are infected with this organism. Thus, routine serologic testing for antibody to T. cruzi may be warranted in immigrants from these countries, especially in view of the potentially serious consequences of infection with this parasite, and also because of the risk of transmission of T. cruzi by blood transfusion.

Interruption of a Trypanosoma cruzi gene encoding a protein containing 14-amino acid repeats by targeted insertion of the neomycin phosphotransferase gene.

In Trypanosoma cruzi, the cause of Chagas' disease in Latin America, a large proportion of the antigenic proteins described to date have repetitive domains. In earlier work we identified a partial length cDNA, designated TCR27, encoding approx. 26 copies of a 14-amino acid repeat and a unique 61-amino acid C-terminal region. The goal of the current project was to replace the repetitive region of a TCR27 gene with the neomycin phosphotransferase gene (NEOr). A pBluescript-based vector was constructed in which the 0.9-kb NEOr coding region replaced the 2.9-kb internal repetitive segment of a TCR27 gene and was in frame with its nonrepetitive 5' coding sequence (pTCR27-2::NEO). Epimastigotes were electroporated in the presence of linearized pTCR27-2::NEO and transfected clones were selected on solid medium containing G418. Southern and Northern analyses of DNAs and RNAs from four G418-resistant clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by NEOr. The absence of the native TCR27-2 protein in the transfected clones was confirmed by Western blot. In axenic cultures growth rates of epimastigotes bearing an interrupted TCR27-2 gene were not different from those of wild-type parasites. In addition, there was no relative impairment of the four transfected clones' ability to proliferate in cultured mammalian cells. The fact that the clones having the interrupted TCR27-2 gene were not impaired biologically suggests that the length of the repetitive region of the TCR27 protein is not a critical factor for survival.

Strains and clones of Trypanosoma cruzi differ in their expression of a surface antigen identified by a monoclonal antibody.

Four Trypanosoma cruzi strains and 50 clones derived from a total of 10 strains were assayed with a monoclonal antibody, WIC 29.26 Ab, for expression of an epitope previously demonstrated to be on the carbohydrate portion of a 72 000 Da surface glycoprotein of Y strain epimastigotes. WIC 29.26 Ab bound to only 2 of the 4 strains and 23 of the 50 clones tested. A group of 10 cloned isolates from one strain contained both reactive and non-reactive clones. Competitive inhibition studies with soluble extracts of the reactive and non-reactive isolates suggested that in addition to being absent from the surface membrane, the antigenic determinant is not synthesized by the non-reactive parasites. These data indicate that the epitope recognized by WIC 29.26 Ab is not present on all T. cruzi epimastigotes, and provide the first demonstration of clone-specific differences in a parasite antigen detected by a monoclonal antibody. No correlation was found between the reactivity of isolates with WIC 29.26 Ab and the previously investigated parameters of growth rate and modal volume.

Characterization of kinetoplast DNA minicircles in Trypanosoma rangeli.

Kinetoplast DNA, the mitochondrial DNA of kinetoplastid protozoans, is a network of interlocked minicircles and maxicircles. We analyzed the sequence organization of minicircle DNAs in the El Tocuyo strain and the San Augustin clone B6 of Trypanosoma rangeli. The frequencies of different minicircle types, as defined by the number of 136-bp conserved regions (CRs), are different in the two strains. About half of the 1.7-kb T. rangeli El Tocuyo minicircles have 1 CR and most of the others have 2. In contrast, most of the 1.6-kb T. rangeli San Augustin minicircles have 2 CRs, while some have four. The CR contains a replication origin at one end and is conserved both within and between the two strains. Comparisons of the T. rangeli El Tocuyo and T. rangeli San Augustin minicircle CRs with minicircle CRs of other kinetoplastid species reveal that they are most similar to those of Trypanosoma cruzi.

Cell cycle expression of histone genes in Trypanosoma cruzi.

In yeast and mammalian cells, the cell cycle-dependent histone genes are typically expressed at a 15- to 35-fold higher level during S phase than during other phases of the cell cycle due to increases in both their transcription rates (three- to 17-fold) and the stabilities of their mRNAs (three to fivefold). In the protozoan trypanosomatids, most life cycle stage-specific genes are not regulated by changes in transcription rates, but are controlled entirely by post-transcriptional events. In contrast, little is known about cell cycle-dependent regulation of trypanosomatid genes. To examine cell cycle-associated expression of histone genes in a trypanosomatid, Trypanosoma cruzi epimastigotes were synchronized with hydroxyurea. The steady state levels of histone mRNAs in the G1, S and G2 phases of the cell cycle were found to vary only two- to fourfold, peaking in S phase. Nuclear run on assays showed that the histone genes are transcribed by RNA polymerase II and that their transcription rates do not increase in S phase relative to G1 and G2. Thus, during S phase of T. cruzi the increase in histone mRNA stability is about the same as in mammals and yeast, but no corresponding increase in the transcription rates of the histone genes occurs.

Trypanosoma cruzi exhibits inter- and intra-strain heterogeneity in molecular karyotype and chromosomal gene location.

Molecular karyotypes of 6 strains and 6 clones of Trypanosoma cruzi were determined using orthogonal-field-alternation gel electrophoresis. At least 15 different chromosome-sized DNA molecules, ranging in size from less than 200 kilobase pairs to greater than 2000 kilobase pairs, were resolved for each of the isolates examined. Many of the bands were present in different relative intensities suggesting that the number of individual chromosomes per organism may be considerably higher. Significant inter- and intra-strain differences in molecular karyotype and in the chromosomal locations of the genes for the spliced leader, tubulins, 5S ribosomal RNA and a heat shock protein were found. These marked chromosomal differences among T. cruzi strains and clones may be related to the high degree of phenotypic heterogeneity previously found in this parasite.

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.

Characterization of a Trypanosoma cruzi poly(A)-binding protein and its genes.

We have characterized the biochemical properties of a 66-kDa poly(A)-binding protein (PABP1) in the protozoan Trypanosoma cruzi and isolated two classes of cDNAs encoding the protein. In concordance, Southern blots showed the presence of 2 gene copies. The two cDNA classes differ in the length of adenosine-rich segments in the 5' untranslated region and in point changes scattered throughout the sequence, but their 1650-bp open reading frames encode identical proteins. A single mRNA of 5.5 kb was detected, indicating that the noncoding regions are unusually long. Both the mRNA and the protein are constitutively expressed in all stages of T. cruzi life cycle. The biochemical properties and sequence comparisons show that the T. cruzi PABP1 is similar to the PABP1 of other eukaryotic organisms. These results indicate that PABP1 has been conserved throughout eukaryotic evolution.

Chagas disease. American trypanosomiasis.

Chagas disease, caused by the protozoan parasite, Trypanosoma cruzi, is a major source of morbidity and death in Latin America. Many infected immigrants from that region now reside in the United States, posing a risk of transfusion-associated transmission of the organism. Serologic testing is the cornerstone of diagnosing chronic T. cruzi infections, and improved assays are needed. Drug treatment is problematic because the two available drugs can have severe side effects and lack efficacy. T. cruzi infection can be particularly severe in immunosuppressed patients.

A case of schistosomiasis japonica: resolution of CAT-scan detected cerebral abnormalities without specific therapy.

A 28-year-old Marine Corps officer developed Katayama fever with central nervous system (CNS) manifestations 6 weeks after swimming on Leyte Island in the Philippines. Symptoms consisted of fever, nausea and vomiting, focal visual field deficits and mild confusion. CAT-scan of the patient's head initially revealed multiple lucencies and severe edema in the left frontal, parietal and occipital lobes. No schistosome eggs were found in the patient's stool, and therefore he was treated with a 10-week course of dexamethasone with resolution of all symptoms over 3 months. Repeat CAT-scan after symptoms cleared showed complete resolution of the focal abnormalities seen earlier. The diagnosis was subsequently established by positive serology and by finding eggs in the patient's stool and in tissue obtained by liver biopsy. This is the first report of CAT-scan-detected focal CNS lesions in a patient with acute schistosomiasis japonica, and resolution of the CNS abnormalities, temporally related to non-specific steroid treatment, is documented as well.

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.

Asymptomatic perianal shedding of herpes simplex virus in patients with acquired immunodeficiency syndrome.

OBJECTIVE: To determine the frequency of asymptomatic perianal shedding of herpes simplex virus (HSV) in adult patients with acquired immunodeficiency syndrome (AIDS). DESIGN: Cross-sectional study. SETTING: A 1000-bed, state-supported hospital in Brazil that provides comprehensive health care. PATIENTS: Eighty-two consecutively hospitalized patients with AIDS (Centers for Disease Control and Prevention class C). MAIN OUTCOME MEASUREMENT: Specimens for HSV culture were obtained with premoistened swabs of the perianal region at approximately 7-day intervals during the hospitalization of each patient. After the specimens were inoculated into cultures of human foreskin and Vero cells, supernatants of cultures showing the cytopathic effect characteristic of HSV infection were tested for virus in a confirmatory immunoenzymatic assay. Typing of HSV was performed by polymerase chain reaction amplification of HSV-1- and HSV-2-specific DNA polymerase sequences. RESULTS: On early into the study, 12 (15%) of 82 patients had perianal ulceration and 70 did not. None of the patients in the latter group developed perianal ulcers during the study period, but HSV was isolated at least once from 17 (24%) of them. Nine of the 17 asymptomatic perianal shedders had a mean of 3 perianal swabs collected before the first HSV isolation, and 11 (65%) of 17 had a total of 18 perianal swabs collected 8 to 62 days after the HSV isolation. All postpositive samples were negative for HSV except 1 obtained from a patient 13 days after the first positive sample. Twelve of the 17 asymptomatic perianal shedders of HSV were followed up clinically for 8 to 62 days after the first episode of shedding and none developed perianal ulceration. CONCLUSIONS: We conclude that asymptomatic perianal shedding of HSV is common in patients with AIDS, even among those without a history of perianal HSV lesions. This shedding appears to be short-lived, intermittent, and not associated with early subsequent development of perianal ulcers. These findings present a new perspective on the natural course of perianal HSV infection in patients with AIDS.

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.

Repetitive protein antigens of Trypanosoma cruzi have diverse intracellular locations.

We previously identified by immunoscreening Trypanosoma cruzi cDNA libraries a group of proteins containing long stretches of tandem repeats. The goal of the current project was to gain insight into the functions of these proteins through ultrastructural analyses consisting of western blotting and electron microscopic localization studies. By comparing western blots of total parasite lysate and different fractions of T. cruzi, we found that 3 of the repetitive antigens are exclusively associated with the parasite membrane, or cytoskeleton, or both. One of the 4 repetitive antigens studied has some association with the membrane or cytoskeleton but also appears to be free in the cytosol. In immunoelectron microscopic studies, the 4 repetitive antigens were detected in different intracellular locations. One of the proteins is located between the flagellum and parasite body, the second has a nuclear distribution, the third is associated with the cell membrane, and the fourth is dispersed throughout the cytoskeletal network. These findings suggest that despite the general structure similarities of these repetitive proteins, they may serve different cellular functions.

Trypanosoma cruzi infection in Walker hounds from Virginia.

Trypanosomiasis has been reported in dogs from Texas, Oklahoma, Louisiana, and South Carolina. We describe the first isolation and characterization of Trypanosoma cruzi from a Walker Hound pup in Virginia that also had postvaccinal distemper. The mother of the pup and 7 of its 8 siblings also were found to be infected with T cruzi, suggesting that the parasite had been transmitted transplacentally or through lactation. Parasitologic, serologic, histologic, and molecular methods were used to establish the diagnosis of T cruzi infection in these dogs. In a serologic survey of 12 dogs (including the sire of the pups) from the area in which the index case occurred, none were found to have antibodies to T cruzi. However, 2 of a further 52 dogs from different areas (to the index case), but in the same county, were seropositive to T cruzi. These findings indicate that canine trypanosomiasis is present in an area of the United States not previously known to be enzootic.

Prevalence of American trypanosomiasis (Chagas disease) among dogs in Oklahoma.

OBJECTIVE: To determine the prevalence of Trypanosoma cruzi infection among dogs in Oklahoma. DESIGN: Cross-sectional study. ANIMALS: 301 owned or impounded dogs related by ownership or general geographic location to 3 dogs determined to have trypanosomiasis. PROCEDURES: Blood samples were obtained from dogs between November 1996 and September 1997. Infection status was determined by use of a radioimmunoprecipitation assay. Second blood samples were obtained from some of the seropositive dogs for study by hemoculture and polymerase chain reaction (PCR) assay. Sites where infected dogs were found were inspected for triatomine insects, and light traps were used for vector trapping. RESULTS: 11(3.6%) dogs were seropositive for T. cruzi infection. Ten of the 11 were owned rural hunting dogs. Protozoal organisms isolated from the blood of 1 seropositive dog were identified as T. cruzi by PCR testing. Only 1 adult Triatoma sanguisuga was captured in a light trap at a site near infected dogs; this insect was not infected. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that T. cruzi is enzootic in eastern Oklahoma. Measures that would reduce the risk of dogs acquiring T. cruzi infection are unlikely to be acceptable to their owners, and no effective drugs are available for treatment. The presence of T. cruzi-infected dogs poses a threat of transmission to persons at risk of exposure to contaminated blood Veterinarians who practice in the southern United States should be cognizant of this blood borne zoonosis and educate all personnel about appropriate precautions.

Use of a PCR assay for diagnosing African trypanosomiasis of the CNS: a case report.

The diagnosis of African trypanosomiasis is parasitologic and often can be difficult, especially in patients infected with Trypanosoma brucei gambiense, the cause of West African sleeping sickness. In the United States imported cases of sleeping sickness are rare, and most occur in tourists returning from East African game parks rather than among immigrants. I report here the use of a T. brucei specific PCR assay in a West African immigrant who presented with neurological symptoms more than 12 years after he had last been in Africa. The patient's historical and physical findings, as well as abnormal cerebrospinal fluid (CSF) parameters, suggested a diagnosis of sleeping sickness. The diagnosis was confirmed when the PCR assay demonstrated the presence of parasite DNA in CSF and blood. Several months after curative therapy the CSF continued to be positive by PCR. These findings suggest that the PCR assay may be useful for sensitive and specific diagnosis of sleeping sickness, but that it may not be helpful for assessing the effect of drug treatment.