A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.

Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

IntroductionMicrosporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization.MethodsLarvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles.ResultsThe molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon.ConclusionsSamples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.

Friedreich's ataxia: clinical and molecular study of 25 Brazilian cases

INTRODUCTION: Friedreich's ataxia is a neurodegenerative disorder whose clinical diagnostic criteria for typical cases basically include: a) early age of onset (< 20 or 25 years), b) autosomal recessive inheritance, c) progressive ataxia of limbs and gait, and d) absence of lower limb tendon reflexes. METHODS: We studied the frequency and the size of expanded GAA and their influence on neurologic findings, age at onset, and disease progression in 25 Brazilian patients with clinical diagnosis of Friedreich's ataxia - 19 typical and 6 atypical - using a long-range PCR test. RESULTS: Abnormalities in cerebellar signs, in electrocardiography, and pes cavus occurred more frequently in typical cases; however, plantar response and speech were more frequently normal in this group when the both typical and atypical cases were compared. Homozygous GAA expansion repeats were detected in 17 cases (68%) - all typical cases. In 8 patients (32%) (6 atypical and 2 typical), no expansion was observed, ruling out the diagnosis of Friedreich's ataxia. In cases with GAA expansions, foot deformity, cardiac abnormalities, and some neurologic findings occurred more frequently; however, abnormalities in cranial nerves and in tomographic findings were detected less frequently than in patients without GAA expansions. DISCUSSION: Molecular analysis was imperative for the diagnosis of Friedreich's ataxia, not only for typical cases but also for atypical ones. There was no genotype-phenotype correlation. Diagnosis based only on clinical findings is limited; however, it aids in better screening for suspected cases that should be tested. Evaluation for vitamin E deficiency is recommended, especially in cases without GAA expansion.

The Amazonia Variant of Vibrio cholerae: Molecular Identification and Study of Virulence Genes

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the El Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

Molecular characterisation of intermediate snail hosts and the search for resistance genes

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a "total evidence" approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.

Molecular diversity of serial Cryptococcus neoformans isolates from AIDS patients in the city of São Paulo, Brazil

Despite highly active anti-retroviral therapy, cryptococcal meningoencephalitis is the second most prevalent neurological disease in Brazilian AIDS patients, being frequently a defining condition with several episodes. As knowledge of Cryptococcus neoformans isolates in the same episode is critical for understanding why some patients develop several episodes, we investigated the genotype characteristics of C. neoformans isolates in two different situations. By pulsed field gel electrophoresis and random amplifield polymorphic DNA analysis, 54 isolates from 12 patients with AIDS and cryptococcosis were analyzed. Group 1 comprised 39 isolates from nine patients with a single episode and hospitalization. Group 2 comprised 15 isolates from three patients with two episodes and hospitalizations. Except for three patients from group 1 probably infected with a single C. neoformans isolate, the other nine patients probably were infected with multiple isolates selected in different collection periods, or the infecting isolate might have underwent mutation to adapt and survive the host immune system and/or the antifungal therapy. However, the three patients from group 2 presented genetic diversity among isolates collected in both hospitalizations, possibly having hosted the initial isolate in both periods. These data, emphasize that Cryptococcus diversity in infection can contribute to strategies of treatment and prevention of cryptococcosis.

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.

Yield QTL analysis of Oryza sativa x O. glumaepatula introgression lines

The objective of this work was to evaluate the yield performance of two generations (BC2F2 and BC2F9) of introgression lines developed from the interspecific cross between Oryza sativa and O. glumaepatula, and to identify the SSR markers associated to yield. The wild accession RS‑16 (O. glumaepatula) was used as donor parent in the backcross with the high yielding cultivar Cica‑8 (O. sativa). A set of 114 BC2F1 introgression lines was genotyped with 141 polymorphic SSR loci distributed across the whole rice genome. Molecular analysis showed that in average 22% of the O. glumaepatula genome was introgressed into BC2F1 generation. Nine BC2F9 introgression lines had a significantly higher yield than the genitor Cica‑8, thus showing a positive genome interaction among cultivated rice and the wild O. glumaepatula. Seven QTL were identified in the overall BC2F2, with one marker interval (4879‑EST20) of great effect on yield. The alleles with positive effect on yield came from the cultivated parent Cica‑8.

Agronomic and molecular traits of the 'Italia Muscat' clone in Sao Francisco River Valley

The objective of this study was to evaluate agronomic and molecular traits of the 'Italia Muscat' clone and compare it with the cv. 'Italia', providing information to support the cultivation of 'Italia Muscat' this cultivar in the São Francisco River Valley. Agronomic characteristics of both clones were evaluated for two seasons in 2004. The characteristics were phenology, bud break (%), bud fertility (%), yield (kg) mass of bunches (g), length and width of bunches (cm), mass of berries (g), length and diameter of berries (mm), TSS (ºBrix), ATT (% titratable acidity) and TSS/TTA. Molecular analysis of seven SSR markers were carried out. The clone 'Italia Muscat' showed larger berries, mass of bunches and better TSS/TA ratio than 'Italia'. The molecular analysis resulted in the same allelic profile in both clones, highlighting the need to use a larger number of microsatellite markers or other molecular technique to allow their discrimination. Based on their morpho-agronomic characteristics, 'Italia Muscat' seems to be a good table grape cultivar alternative for grape growers of São Francisco River Valley.

Introdução a modelagem molecular de fármacos no curso experimental de química farmacêutica

Molecular Modeling is an important tool in drug design and it is very useful to predict biological activity from a library of compounds. A wide variety of computer programs and methods have been developed to visualize the tridimensional geometry and calculate physical properties of drugs. In this work, we describe a practical approach of molecular modeling as a powerful tool to study structure-activity relationships of drugs, including some antibacterials, hormones, cholinergic and adrenergic agents. At first, the students learn how to draw 3D structures and use them to perform conformational and molecular analysis. Thus, they compare drugs with similar pharmacological activity by superimposing one structure on the top of another and evaluate the geometry and physical properties.

Occurrence and molecular characterization of cryptococcosis in dogs and cats in Mato Grosso, Brazil

Cryptococcosis is an infection that affects humans and animals, the etiology is attributed to Cryptococcus neoformans variety neoformans, C. neoformans var. grubii and Cryptococcus gattii. The infection is common in dogs and cats, causing respiratory, neurological, cutaneous and ocular infections. Aiming to better understand the epidemiology of cryptococcosis in animals in the region, this paper describe the occurrence and characterization of the Cryptococcus species involved in this illness in pet animals at Mato Grosso State, Brazil. Clinical samples of four cases, two in cats and two dogs, were submitted for pathological, microbiological and molecular analysis. Microscopically, in three cases, tissue sections stained with hematoxylin and eosin had absence to severe granulomatous reaction composed by histiocytes, multinucleated cells and lymphocytes infiltration. In one case, citological imprint analysis showed similar inflammatory mainly mononuclear and lymphocyte cells infiltration. All cases had variable amounts of intracellular and extracellular fungal structures compatible with Cryptococcus sp. on Periodic Acid-Schiff (PAS) stain. All clinical samples were positive for culture on Sabouraud Dextrose Agar (SDA) and morphologically classified as Cryptococcus sp. The isolates were PCR positive for C. gatti, being confirmed by sequencing technique. The findings characterize the molecular species involved in animal infections in the region, and may contribute to future studies of the epidemiology of C. gattii.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

Genomic analysis of Brazilian patients with Fabry disease

Fabry disease is an X-linked lysosomal disorder due to a-galactosidase A deficiency that causes storage of globotriaosylceramide. The gene coding for this lysosomal enzyme is located on the long arm of the X chromosome, in region Xq21.33-Xq22. Disease progression leads to vascular disease secondary to involvement of kidney, heart and the central nervous system. Detection of female carriers based solely on enzyme assays is often inconclusive. Therefore, mutation analysis is a valuable tool for diagnosis and genetic counseling. Many mutations of the a-galactosidase A gene have been reported with high genetic heterogeneity, being most mutations private found in only one family. The disease is panethnic, and estimates of incidence range from about 1 in 40,000 to 60,000 males. Our objective was to describe the analysis of 6 male and 7 female individuals belonging to 4 different Fabry disease families by automated sequencing of the seven exons of the a-galactosidase gene. Sequencing was performed using PCR fragments for each exon amplified from DNA extracted from peripheral blood. Three known mutations and one previously described in another Brazilian family were detected. Of 7 female relatives studied, 4 were carriers. Although the present study confirms the heterogeneity of mutations in Fabry disease, the finding of the same mutation previously detected in another Fabry family from our region raises the possibility of some founder effect, or genetic drift. Finally, the present study highlights the importance of molecular analysis for carrier detection and genetic counseling.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.