Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.

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Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament.

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.

First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis). The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH) was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%), C. tropicalis (23%), C. glabrata (14%) and C. krusei (4%). This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

Recent advances in the diagnosis of Schistosoma infection: the detection of parasite DNA

As Schistosoma sp. control programs are chiefly based on treatment of infected population, adequate case finding has a crucial role. The available diagnostic methods are far from ideal, since the search for eggs in stools and the detection of circulating antigens lack sensitivity in low prevalence and post-treatment situations and antibody detection lacks specificity. In most endemic foci, repeated treatment of infected people leaves a number of non-diagnosed and consequently non-treated persons, enough to maintain a persistent residue of 5 to 10% prevalence. In an attempt to surpass these diagnostic limitations we have developed a polymerase chain reaction (PCR) for the detection of Schistosoma sp. in feces that, in a first population study, has shown to be more sensitive than three-repeated stool Kato-Katz examination. The PCR may constitute a valuable tool for the diagnosis of the Schistosoma sp. infection in special situations, when high sensitivity and specificity are required and infrastructure is available.

A novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi

We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions.

Report of the Vaccine Damage Steering Group

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The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.

A long, naturally presented immunodominant epitope from NY-ESO-1 tumor antigen: implications for cancer vaccine design.

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.

Persistent human parvovirus B19 infection in children under maintenance chemotherapy for acute lymphocytic leukemia.

OBJECTIVE: To report on B19 infection management and chemotherapy schedule consequences in five children treated for acute lymphocytic leukemia (ALL). PATIENTS AND METHODS: Between May 2001 and February 2002, five patients between 4 and 12 years of age, receiving maintenance chemotherapy for ALL, presented with symptoms suggesting B19 infection (pallor, fatigue, petechiae and pancytopenia in four patients; generalized rash in two patients; acute hepatitis in one patient). Qualitative polymerase chain reaction (PCR) on peripheral blood was used for diagnosis and follow-up of infection; quantitative PCR was used for viral load measurement. Intravenous nonspecific high-dose immunoglobulin therapy was administered until PCR was negative. RESULTS: Qualitative B19 DNA was found in the peripheral blood of all patients, confirming the infection. Viral load at diagnosis ranged from 10 to 10 particles/mL blood. B19 DNA was detectable in four patients at 45, 21, 40, and 44 weeks, respectively. Chemotherapy was delayed in all patients. No clear benefit of intravenous immunoglobulin was noted. CONCLUSIONS: Infection with B19 is rarely reported in patients with ALL, but it should be suspected when unexplained pancytopenia occurs during chemotherapy. Persistent B19 infection remains a challenge in the management of patients receiving maintenance chemotherapy for ALL, as no specific therapy such as a specific immunoglobulin or vaccine exists. The role of viral load measurement needs to be established in terms of its use in follow-up and evaluation of the therapeutic response.

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and Northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001). The variation among individuals within populations (phiST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Prevalence of human papillomavirus types in women with pre-neoplastic and neoplastic cervical lesions in the Federal District of Brazil

As a contribution to the public health authorities in planning prophylactic and therapeutic vaccine strategies, we describe the prevalence of human papillomavirus (HPV) types in women presenting abnormal cytological results in Pap smear screening tests in the Federal District, Central Brazil. We studied 129 cervical scraping samples from women whose cytological tests showed either pre-neoplastic or neoplastic lesions. Amplification of HPV DNA was performed by polymerase chain reaction using consensus primers MY09 and MY11 followed by identification of isolates by restriction fragment length polymorphism. We detected HPV DNA in 62% of the samples, including HPV-16 in 43.8%, HPV-58 in 12.5%, HPV-31 in 10%, HPV-53 in 6.3%, each of HPV-18 and HPV-33 in 3.8% of the isolates. Other types (HPV-35, -52, -66, -CP8304, -6, -11, and -CP8061) were less frequent (= or < 2.5% each). The prevalence of HPV-58 was relatively higher in this population than in data in South America, but similar to results obtained in other studies in Latin America, Europe, and Eastern Asia. Case-control studies need to be carried out to establish the association between the prevalence of HPV types specially the less frequent high-risk types and cervical cancer.

Preliminary results of random amplification of polymorphic DNA among Triatominae of the phyllosoma complex (Hemiptera, Reduviidae)

In Mexico, Triatoma longipennis (Usinger), Triatoma picturata (Usinger), and Triatoma pallidipennis (Stal), primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD). Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug) and Triatoma barberi (Usinger). Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.