Multiplex real-time PCR for the diagnosis of malaria: correlation with microscopy.

Malaria is generally diagnosed by microscopy and rapid antigen testing. Molecular methods become more widely used. In the present study, the contribution of a quantitative multiplex malaria PCR was investigated. We assessed: (i) the agreement between PCR-based identification and microscopy and (ii) the correlation between the parasite load as determined by quantitative PCR and by microscopy. For 83 patients positive by microscopy for Plasmodium spp., the first EDTA-blood sample was tested by multiplex PCR to confirm smear-based species identification. Parasite load was assessed daily using both microscopy and PCR. Among the 83 patients tested, one was positive by microscopy only and 82 were positive by microscopy and PCR. Agreement between microscopy and PCR for the identification at the species level was 89% (73/82). Six of the nine discordant results corresponded to co-infections by two or three species and were attributed to inaccurate morphological identification of mixed cases. The parasite load generally decreased rapidly after treatment had been started, with similar decay curves being obtained using both microscopy and PCR. Our PCR proved especially useful for identifying mixed infections. The quantification obtained by PCR closely correlated with microscopy-based quantification and could be useful for monitoring treatment efficacy, at least in clinical trials.

Innovation or Imitation? The effect of spillovers and competitive pressure on firms' R&D strategy choice

This article provides a theoretical and empirical analysis of a firm's optimal R&D strategy choice. In this paper a firm's R&D strategy is assumed to be endogenous and allowed to depend on both internal firms. characteristics and external factors. Firms choose between two strategies, either they engage in R&D or abstain from own R&D and imitate the outcomes of innovators. In the theoretical model this yields three types of equilibria in which either all firms innovate, some firms innovate and others imitate, or no firm innovates. Firms'equilibrium strategies crucially depend on external factors. We find that the efficiency of intellectual property rights protection positively affects firms'incentives to engage in R&D, while competitive pressure has a negative effect. In addition, smaller firms are found to be more likely to become imitators when the product is homogeneous and the level of spillovers is high. These results are supported by empirical evidence for German .rms from manufacturing and services sectors. Regarding social welfare our results indicate that strengthening intellectual property protection can have an ambiguous effect. In markets characterized by a high rate of innovation a reduction of intellectual property rights protection can discourage innovative performance substantially. However, a reduction of patent protection can also increase social welfare because it may induce imitation. This indicates that policy issues such as the optimal length and breadth of patent protection cannot be resolved without taking into account specific market and firm characteristics. Journal of Economic Literature Classification Numbers: C35, D43, L13, L22, O31. Keywords: Innovation; imitation; spillovers; product differentiation; market competition; intellectual property rights protection.

Desenvolupament d'eines d'alta ressolució per al cribatge (screening) de l'activitat de fàrmacs

Projecte de recerca elaborat a partir d’una estada a la Satandford University, EEUU, entre 2007 i 2009. Els darrers anys, hi ha hagut un avanç espectacular en la tecnologia aplicada a l’anàlisi del genoma i del proteoma (microarrays, PCR quantitativa real time, electroforesis dos dimensions, espectroscòpia de masses, etc.) permetent la resolució de mostres complexes i la detecció quantitativa de diferents gens i proteïnes en un sol experiment. A més a més, la seva importància radica en la capacitat d’identificar potencials dianes terapèutiques i possibles fàrmacs, així com la seva aplicació en el disseny i desenvolupament de noves eines de diagnòstic. L’aplicabilitat de les tècniques actuals, però, està limitada al nivell al que el teixit pot ser disseccionat. Si bé donen valuosa informació sobre expressió de gens i proteïnes implicades en una malaltia o en resposta a un fàrmac per exemple, en cap cas, s’obté una informació in situ ni es pot obtenir informació espacial o una resolució temporal, així com tampoc s’obté informació de sistemes in vivo. L’objectiu d’aquest projecte és desenvolupar i validar un nou microscopi, d’alta resolució, ultrasensible i de fàcil ús, que permeti tant la detecció de metabòlits, gens o proteïnes a la cèl•lula viva en temps real com l’estudi de la seva funció. Obtenint així una descripció detallada de les interaccions entre proteïnes/gens que es donen dins la cèl•lula. Aquest microscopi serà un instrument sensible, selectiu, ràpid, robust, automatitzat i de cost moderat que realitzarà processos de cribatge d’alt rendiment (High throughput screening) genètics, mèdics, químics i farmacèutics (per aplicacions diagnòstiques i de identificació i selecció de compostos actius) de manera més eficient. Per poder realitzar aquest objectius el microscopi farà ús de les més noves tecnologies: 1)la microscopia òptica i d’imatge, per millorar la visualització espaial i la sensibilitat de l’imatge; 2) la utilització de nous mètodes de detecció incloent els més moderns avanços en nanopartícules; 3) la creació de mètodes informàtics per adquirir, emmagatzemar i processar les imatges obtingudes.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Possible competitive displacement of planorbids by Melanoides tuberculata in Minas Gerais, Brazil

Several species of snails, including Pomacea haustrum, Marisa cornuarietis and Helisoma duryi, have been identified as probable competitors and/or predators of planorbid intermediate hosts of Schistosoma. During the last few years, studies carried out in the Caribbean region have shown reductions and even disappearances of populations of Biomphalaria glabrata and B. straminea in breeding places where the snail Melanoides tuberculata was introduced. Observations made over a period of 10 years in two lakes close to Belo Horizonte, MG, showed that there were marked reductions in autochthonous populations of B. glabrata and B. straminea after the arrival of M. tuberculata, both Biomphalaria species disappearing completely after eight years.

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.

High proportion of wrongly identified methicillin-resistant Staphylococcus aureus carriers by use of a rapid commercial PCR assay due to presence of staphylococcal cassette chromosome element lacking the mecA gene.

During a 9-month period, 217 patients were newly diagnosed as methicillin-resistant Staphylococcus aureus (MRSA) carriers by using a commercial rapid PCR-based test (GeneXpert). However, no MRSA was recovered by culturing the second swab in 61 of these patients. Further analyses showed that 28 (12.9%) of the patients harbored S. aureus isolates with a staphylococcal cassette chromosome element lacking the mecA gene and were thus incorrectly determined to be MRSA carriers.

Production of low phytic acid rice by hairpin RNA- and artificial microRNA-mediated silencing of OsMIK in seeds

To produce agronomically competitive rice with nutritionally superior, environmentally safe phytic acid (PA) levels, hairpin RNA (hpRNA)- and artificial microRNA (amiRNA)-mediated gene silencing approaches were explored to reduce both myo-inositol kinase gene (OsMIK) expression and PA accumulation in rice seeds. hpRNA and amiRNA sequences targeted to OsMIK (hpMIK and amiMIK), under the control of a rice Ole18 promoter, were transformed into the rice cultivar Nippon-bare. Fourteen and 21 independent transgenic events were identified containing the hpMIK and amiMIK constructs, respectively, from which five stable homozygous transgenic lines of each were developed together with their null siblings. Southern blotting demonstrated transgene integration into the genome and quantitative real-time PCR showed that gene silencing was restricted to seeds. OsMIK transcripts were significantly reduced in both transgenic amiMIK and hpMIK seeds, which had PA levels reduced by 14.9-50.2 and 38.1-50.7 %, respectively, compared with their respective null siblings. There were no systematic significant differences in agronomic traits between the transgenic lines and their non-transgenic siblings, and no correlation between seed PA contents and decreased rates of seed germination and seedling emergence. The results of the present study suggest that Ole 18-driven OsMIK silencing via hpRNA and amiRNA could be an effective way to develop agronomically competitive low phytic acid rice.

A Multiplex-PCR approach to identification of the Brazilian intermediate hosts of Schistosoma mansoni

Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.

Establishment of HEV RNA reverse transcriptase PCR assays for the diagnosis of acute and chronic hepatitis E

Background and aim: H epatitis E v irus (HEV) infection has emerged as a c ause o f travel-related a nd autochthonous a cute hepatitis as well as chronic hepatitis in immunosuppressed patients. While t ravel-related cases a re c aused primarily b y infections w ith HEV of g enotype 1 ( HEV-1), autochthonous c ases a nd chronic cases a re d ue t o genotype 3 (HEV-3), which is s hared between humans and diverse animal species. The aim of this study was to establish HEV RNA detection assays f or q uantitative v iral load testing and genotyping. Methods: V iral RNA was p urified from plasma or s erum a nd converted to cDNA prior to (1) multiplex real-time PCR for HEV RNA quantification and (2) multiplex PCR coupled to DNA sequencing for HEV genotype determination. Real-time PCR was d esigned to match a ll known HEV genotypes available i n Genbank while PCR was designed using conserved primers flanking a variable region of the HEV RNA. Results: In a validation panel, the newly developed assays allowed for the reliable detection and genotyping of HEV-1 or HEV-3. Cases of t ravel-related and a utochthonous a cute h epatitis E a s well a s chronic hepatitis E i n immunosuppressed patients have b een identified using t hese a ssays a nd will be p resented in detail. Anti- HEV antibodies were n egative i n three well-characterized patients with chronic hepatitis E after organ transplantation. Conclusions: We developed and validated a quantitative HEV RNA detection assay that c an now be o ffered on a r outine basis (www.chuv.ch/imul/imu-collaborations-viral_hepatitis). Genotyping can also be offered on selected cases. HEV RNA detection is key in diagnosing chronic hepatitis E i n immunosuppressed patients with unexplained transaminase elevations, as serology can be negative in these patients.

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

Hepatitis C positive, PCR positive: Clinician results factsheet

Factsheet for clinicians with patients who are antibody positive, and polymerase chain reaction (PCR) positive, and therefore have chronic hepatitis C infection.

Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS) region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

Multiplex-PCR for detection of natural Leishmania infection in Lutzomyia spp. captured in an endemic region for cutaneous leishmaniasis in state of Sucre, Venezuela

We studied the natural infection of Lutzomyia (Lutzomyia) sp. with Leishmania in endemic foci of cutaneous leishmaniasis in the Paria peninsula, state of Sucre, Venezuela. Sand flies were collected between March 2001 and June 2003, using Shannon light-traps and human bait. Of the 1291 insects captured, only two species of phlebotomines were identified: L. ovallesi (82.75%) and L. gomezi (17.42%). A sample of the collected sand flies (51 pools of 2-12 individuals) were analyzed by using a multiplex-PCR assay for simultaneous detection of New Word Leishmaniaand Viannia subgenera. The results showed a total of 8 pools (15.68%) infected; of these, 7 were L. ovallesi naturally infected with L. braziliensis (2 pools) and L. mexicana (5 pools) and 1 pool of L. gomezi infected by L. braziliensis.