Clinical data and molecular analysis of Mycobacterium tuberculosis isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.

Neuropathologie et pathologie moléculaire des gliomes [Neuropathology and molecular pathology of gliomas]

Gliomas are the most frequent primary brain tumours. The WHO classification is essentially based on histological and immunohistochemical criteria. More recently multiple cytogenetic and molecular alterations associated with initiation and progression have been shown and the genetic profiles of tumour entities have been incorporated in the WHO classifiacation. Molecular testing of the MGMT promotor methylation in glioblastoma, predictive for the response to combined radio-/chimiothérapie, and the LOH 1p/19q in oligodendroglial tumours, as prognostic factor supplements the histopathological diagnosis. In the near futur array-based profiling techniques will contribute to a refinement of glioma classification and identify targets for more individualized glioma therapies.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease.

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences.

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

How to keep the brain awake? The complex molecular pharmacogenetics of wake promotion.

Wake-promoting drugs are widely used to treat excessive daytime sleepiness. The neuronal pathways involved in wake promotion are multiple and often not well characterized. We tested d-amphetamine, modafinil, and YKP10A, a novel wake-promoting compound, in three inbred strains of mice. The wake duration induced by YKP10A and d-amphetamine depended similarly on genotype, whereas opposite strain differences were observed after modafinil. Electroencephalogram (EEG) analysis during drug-induced wakefulness revealed a transient approximately 2 Hz slowing of theta oscillations and an increase in beta-2 (20-35 Hz) activity only after YKP10A. Gamma activity (35-60 Hz) was induced by all drugs in a drug- and genotype-dependent manner. Brain transcriptome and clustering analyses indicated that the three drugs have both common and specific molecular signatures. The correlation between specific EEG and gene-expression signatures suggests that the neuronal pathways activated to stay awake vary among drugs and genetic background.

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.

Molecular divergence in the timeless and cpr genes among three sympatric cryptic species of the Anopheles triannulatus complex

Anopheles triannulatus s.l. is a malaria vector with a wide geographic distribution, ranging from Argentina-Nicaragua and Trinidad. Here we analysed sequences of two genes, timeless and cpr, to assess the genetic variability and divergence among three sympatric cryptic species of this complex from Salobra, central-western Brazil. The timeless gene sequences did not conclusively differentiate Anopheles halophylus and An. triannulatus species "C". However, a partial separation has been observed between these species and An. triannulatus s.s. Importantly, the analysis of the cpr gene sequences revealed fixed differences, no shared polymorphisms and considerable genetic differentiation among the three species of the An. triannulatus complex. The results confirm that An. triannulatus s.s., An. halophylus and An. triannulatus species C are distinct taxa, with the latter two likely representing a more recent speciation event.

Application of Molecular Diagnostic Techniques for Viral Testing.

Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.

Lymphoblastic transformation of follicular lymphoma: a clinicopathologic and molecular analysis of 7 patients.

Approximately 30% of patients with follicular lymphoma (FL) transform to a more aggressive malignancy, most commonly diffuse large B cell lymphoma. Rarely, FL transformation results in clinical findings, histology, and immunophenotype reminiscent of B-lymphoblastic leukemia/lymphoma. We report the largest series to date with detailed analysis of 7 such patients. Lymphoblastic transformation occurred on average 2 years after initial diagnosis of FL. Five patients had prior intensive chemotherapy. Two patients developed mature high-grade lymphoma, followed by the lymphoblastic transformation. FL had BCL2 gene rearrangement in 4 of 5 cases. High-grade transformation was accompanied by MYC gene rearrangement (5 of 5). Transformation was characterized by expression of TdT, loss of Bcl6, variable loss of immunoglobulin light chain, and persistence of Pax-5, Bcl2, and CD10. Whole-exome sequencing in 1 case revealed presence of several actionable mutations (CD79B, CCND3, CDK12). FL, aggressive mature B cell lymphoma, and lymphoblastic transformation were clonally related in 6 evaluable cases. After transformation, survival ranged from 1 to 14 months. Four patients died of disease, 2 were in remission after stem cell transplant, and 1 was alive with disease.