Clinical, laboratory and epidemiological aspects of HPV infection in a low-income population from South Bahia, Brazil.

The aim of this study was to determine the prevalence and risk factors for human papillomavirus (HPV) infection in the Southern region of the State of Bahia, evaluating the performance of alternative complementary methods for cervical lesion detection. Cervical samples from women who attended healthcare units were collected and diagnosed by visual inspection, cervical cytology and nested polymerase chain reaction (PCR). Moreover, hemi-nested PCR was performed to detect different HPV genotypes. The prevalence of HPV infection was 47·7%, with genotype 16 detected in most cases. Infection was associated with dyspareunia and bleeding (P < 0·001, odds ratio (OR) 5·6, 95% confidence interval (CI) 2·815-11·14) and hormonal contraceptive use (P = 0·007, OR 2·33, 95% CI 1·25-4·34). There was a positive correlation between positive PCR and positive visual inspection, cervical cytology and symptoms reported. Furthermore, visual inspection was twice as specific, and had a greater positive predictive value than cytology. We showed a high prevalence of HPV infection in Southern Bahia, with HPV 16 being the most common type, and visual inspection being most effective at detecting HPV lesions, corroborating the suggestion that it can be applied in routine gynecologic examinations for low-income populations.

Comparative study of the presence of Trypanosoma cruzi kDNA, inflammation and denervation in chagasic patients with and without megaesophagus.

Neuronal lesions have been considered the hallmark of chagasic megaesophagus, but the role of Trypanosoma cruzi and the participation of the inflammatory cells in this process are still debated. In the present study we counted neurons in the oesophagus from patients with and without megaesophagus and further examined these samples for the presence of parasite kDNA and cells with cytolytic potential (Natural Killer cells, cytotoxic lymphocytes and macrophages). The presence of parasite kDNA was demonstrated in 100% of cases with megaesophagus and in 60% of patients without megaesophagus. When analysed for the number of neurons, the patients without megaesophagus could be classified into 2 groups, as having normal or a decreased number of neurons. The former group did not show any inflammatory process, but interestingly, all patients without megaesophagus presenting decreased number of neurons also presented both parasite kDNA and inflammatory process in the organ. We further observed that the numbers of cytotoxic cells in the myenteric plexus region inversely correlate with the number of neurons. These data together strongly suggest that chronic lesions in chagasic megaesophagus might be a consequence of immune-mediated mechanisms, that last until the chronic phase of infection, and are dependent on the persistence of parasite in the host's tissue.

Avian malaria in Brazilian passerine birds: parasitism detected by nested PCR using DNA from stained blood smears.

The microscopical examination of Giemsa-stained thin blood smears and a nested PCR were performed to detect avian Plasmodium in 275 passerine birds from small and large fragments of Atlantic Forest, Minas Gerais, Brazil. The 275 blood smears were used both for the microscopical examination and nested PCR providing the DNA template used for the reactions. The sensitivity of the nested PCR assay was higher than that observed for blood smears through microscopical examination. High prevalence (39.6%) of Plasmodium infections was detected by nested PCR while the microscopical examination detected only 16.5 % positive birds. Poor agreement was observed between the results of the two different tests. The PCR data obtained were correlated to the forest fragment size of the Atlantic Forest and also correlated to the biological characteristics of the birds (nest type construction, diet, participation in mixed-species flocks, age and sex). Birds captured in the large forest areas were more infected than birds captured in the small areas (51.9 % and 28.5 %, respectively). Diet and participation in mixed-species flocks were correlated to the Plasmodium parasitism. The insectivorous birds and those that participated in mixed-species flocks were more frequently infected (47% and 41.5%, respectively) than the other groups.

Genetic characterization of Trypanosoma cruzi directly from tissues of patients with chronic Chagas disease: differential distribution of genetic types into diverse organs.

We have previously shown that a low-stringency single-specific primer-polymerase chain reaction (LSSP- PCR) is a highly sensitive and reproducible technique for the genetic profiling of Trypanosoma cruzi parasites directly in tissues from infected animals and humans. By applying LSSP-PCR to the study of the variable region of kinetoplast minicircle from T. cruzi, the intraspecific polymorphism of the kinetoplast-deoxyribonucleic acid (kDNA) sequence can be translated into individual kDNA signatures. In the present article, we report on our success using the LSSP-PCR technique in profiling the T. cruzi parasites present in the hearts of 13 patients with chagasic cardiopathy and in the esophagi of four patients (three of them with chagasic megaesophagus). In two patients, one with the cardiodigestive clinical form of Chagas disease and the other with cardiopathy and an esophageal inflammatory process, we could study both heart and esophagus and we detected distinct kDNA signatures in the two organs. This provides evidence of a differential tissue distribution of genetically diverse T. cruzi populations in chronic Chagas disease, suggesting that the genetic variability of the parasite is one of the determining factors of the clinical form of the disease.

Trypanosoma cruzi: optimization of polymerase chain reaction for detection in human blood.

We have optimized the conditions for DNA extraction and polymerase chain reaction (PCR) amplification to diagnose the presence of Trypanosoma cruzi DNA in the blood and serum of patients with chronic Chagas disease. The approximately 330-bp fragment of the kinetoplast minicircles was used as a target for amplification. The use of chemiluminescence on slot blots with a specific alkaline phosphatase-conjugated oligonucleotide probe detected specific product from as little as 0.1 fg of T. cruzi kDNA. An additional product of approximately 200 bp inadvertently amplified from the human genome was observed in human blood from T. cruzi-negative and -positive samples and served as an internal control of the amplification. Samples from other mammalian hosts were also assayed using the PCR protocol. The higher sensitivity of our PCR method observed in both acute and chronic phases of T.cruzi infections in mice and dog, respectively, could be useful in monitoring the course of infection during experimental drug tests in laboratory animals. Since this procedure showed a higher sensitivity than other protocols in the literature, it may be a suitable routine test in diagnosing Chagas disease, especially for patients presenting very low parasitemia levels.

LSSP-PCR for characterization of strains of Entamoeba histolytica isolated in Brazil.

Strains of Entamoeba histolytica isolated in Brazil were characterized using the Low-Stringency Single Specific Primer PCR (LSSP-PCR), that detects single or multiple mutations in gene size DNA fragments. Using this technique, a 482-bp genomic DNA fragment from a structural gene in 8 strains and 2 clones of E. histolytica, isolated from symptomatic and asymptomatic patients in Brazil, including pathogenic and non-pathogenic zymodemes were studied. The results obtained indicate that LSSP-PCR is a valuable method for differentiating strains of E. histolytica. Moreover, the results are consistent with the concept that pathogenic and non-pathogenic strains of E. histolytica may represent distinct species or subspecies and are in accord with phenotypically characteristic isoenzyme patterns.

Kinetoplast DNA signatures of Trypanosoma cruzi strains obtained directly from infected tissues.

We report here a polymerase chain reaction (PCR)-based DNA profiling technique that permits Trypanosoma cruzi strain characterization by direct study of infected tissues. This is based on application of a recently developed method of DNA fragment identification, called low-stringency single specific primer PCR (LSSP-PCR), to the study of the variable region of kinetoplast DNA (kDNA) minicircles from T. cruzi Thus, we can translate the intraspecific polymorphism in the nucleotide sequence of kDNA minicircles into a specific and highly reproducible kDNA signature. Comparison with the phenogram obtained by DNA fingerprinting analysis of a set of T. cruzi strains showed good qualitative correlation between the degree of divergence of the LSSP-PCR profiles and the genetic distance between the strains. kDNA signatures of heart tissue from acutely or chronically infected animals revealed perfect concordance with the patterns obtained from cultured parasites for the CL and Colombiana strains but not for the Y strain, which is known to be multiclonal. However, the match was perfect for studies with two clones of the Y strain. We take this as evidence that in some multiclonal strains there is heterogeneity among the clones in the degree of tropism for the heart tissue. Finally, we showed that it is possible to obtain a T. cruzi kDNA signature from the heart of a human patient with chronic Chagasic myocardiopathy. kDNA signatures obtained by LSSP-PCR of sequences amplified from infected tissues constitute a new tool to study the molecular epidemiology of Chagas' disease.

Mitochondrial D-loop "signatures" produced by low-stringency single specific primer PCR constitute a simple comparative human identity test.

We have developed a technique called "LSSP-PCR" (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique "gene signature." We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing.

Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA.