Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood and nasal secretion of Brazilian household contacts

DNA samples from blood and nasal swabs of 125 healthy household contacts was submitted to amplification by polymerase chain reaction (PCR) using a Mycobacterium leprae-specific sequence as a target for the detection of subclinical infection with M. leprae.All samples were submitted to hybridization analysis in order to exclude any false positive or negative results. Two positive samples were confirmed from blood out of 119 (1.7%) and two positive samples from nasal secretion out of 120 (1.7%). The analysis of the families with positive individuals showed that 2.5% (n = 3) of the contacts were relatives of multibacilary patients while 0.8% of the cases (n = 1) had a paucibacilary as an index case. All positive contacts were followed up and after one year none of them presented clinical signs of the disease. In spite of the PCR sensitivity to detect the presence of the M. leprae in a subclinical stage, this molecular approach did not seem to be a valuable tool to screen household contacts, since we determined a spurious association of the PCR positivity and further development of leprosy.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positivity) by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9), including one obtained by nested-PCR (P-2) and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1%) with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

Ceftriaxone acts synergistically with levofloxacin in experimental meningitis and reduces levofloxacin-induced resistance in penicillin-resistant pneumococci.

Ceftriaxone acted synergistically with levofloxacin in time-killing assays in vitro over 8 h against two penicillin-resistant pneumococcal strains (WB4 and KR4; MIC of penicillin: 4 mg/L). Synergy was confirmed with the chequerboard method, showing FIC indices of 0.25. In the experimental rabbit meningitis model, ceftriaxone (1x 125 mg/kg) was slightly less bactericidal (-0.30 Deltalog(10) cfu/mL(.)h) compared with levofloxacin (-0.45 Deltalog(10) cfu/mL(.)h) against the penicillin-resistant strain WB4. The combination therapy (levofloxacin and ceftriaxone) was significantly superior (-0.64 Deltalog(10) cfu/mL(.)h) to either monotherapy. In cycling experiments in vitro, the addition of ceftriaxone at a sub-MIC concentration (1/16 MIC) reduced levofloxacin-induced resistance in the two strains KR4 and WB4. After 12 cycles with levofloxacin monotherapy, the MIC increased 64-fold in both strains versus a 16-fold increase with the combination (levofloxacin + ceftriaxone 1/16 MIC). In both strains, levofloxacin-induced resistance was confirmed by mutations detected in the genes parC and gyrA, encoding for subunits of topoisomerase IV and gyrase, respectively. The addition of ceftriaxone suppressed mutations in parC but led to a new mutation in parE in both strains.

Detection of Entamoeba histolytica/Entamoeba dispar in stool specimens by using enzyme-linked immunosorbent assay

Entamoeba histolytica actually comprises two genetically distinct but morphologically indistinguishable species. E. histolytica can cause invasive intestinal and extra intestinal disease, while E. dispar cannot. Identification and differentiation of E. dispar and E. histolytica in stool sample by microscopy is imprecise. Several weeks of culture and isoenzyme analysis are required to differentiate E. histolytica from E. dispar. In this study, we have used an enzyme-linked immunosorbent assay (ELISA) for detection of E. histolytica/E.dispar and compared it with microscopy. Eighty-eight samples were evaluated, trichrome staining was positive in 20.4% (18) and ELISA was positive in 29.5% (26). Both tests were positive in 14 (15.9%) samples, 4 (4.5%) only with direct microscopy, and 12 (13.6%) only with ELISA. Both tests were negative in 58 (65.9%) samples. Microscopy has low sensitivity and high specificity, low negative predictive value and high positive predictive value in comparison with ELISA. E. histolytica/E. dispar antigen detection ELISA tests are inexpensive compared to the specific tests, yield objective results and do not require experienced microscopists and can therefore be recommended for screening of stools worldwide and the results can be taken for treatment that are fitting with its clinic.

Dengue virus detection by using reverse transcription-polymerase chain reaction in saliva and progeny of experimentally infected Aedes albopictus from Brazil

Oral susceptibility and vertical transmission of dengue virus type 2 (DENV-2) in an Aedes albopictus sample from Rio de Janeiro was estimated. The infection (36.7%) and transmission (83.3%) rates for Ae. albopictus were higher than those of an Ae. aegypti colony used as control, 32.8 and 60%, respectively. Fourth instar larvae and females descendants of 48.5 and 39.1% of experimentally infected Ae. albopictus showed to harbor the virus. The oral susceptibility and the high capacity to assure vertical transmission exhibited by Ae. albopictus from Brazil reinforce that this species may play a role in the maintenance of the virus in nature and be a threat for dengue control in the country.

Clustering and hot spot detection in socio-economic spatio-temporal data

Distribution of socio-economic features in urban space is an important source of information for land and transportation planning. The metropolization phenomenon has changed the distribution of types of professions in space and has given birth to different spatial patterns that the urban planner must know in order to plan a sustainable city. Such distributions can be discovered by statistical and learning algorithms through different methods. In this paper, an unsupervised classification method and a cluster detection method are discussed and applied to analyze the socio-economic structure of Switzerland. The unsupervised classification method, based on Ward's classification and self-organized maps, is used to classify the municipalities of the country and allows to reduce a highly-dimensional input information to interpret the socio-economic landscape. The cluster detection method, the spatial scan statistics, is used in a more specific manner in order to detect hot spots of certain types of service activities. The method is applied to the distribution services in the agglomeration of Lausanne. Results show the emergence of new centralities and can be analyzed in both transportation and social terms.

Methods for detection and confirmation of Hematide?/peginesatide in anti-doping samples.

Since the 1990's, cheating athletes have abused substances to increase their oxygen transport capabilities; among these substances, recombinant EPO is the most well known. Currently, other investigational pharmaceutical products are able to produce an effect similar to EPO but without having chemical structures related to EPO; these are the synthetic erythropoiesis stimulating agents (ESAs). Peginesatide (also known as Hematide?) is being developed by Affymax and Takeda and, if approved by regulatory authorities, could soon be released on the international market. To detect potential athletic abuse of this product and deter athletes who consider cheating, we initiated a collaboration to implement a detection test for anti-doping purposes. Peginesatide is a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent that is designed and engineered to stimulate specifically the erythropoietin receptor dimer that governs erythropoiesis. It is undetectable using current anti-doping tests due to its lack of sequence homology to EPO. To detect and deter potential abuse of peginesatide, we initiated an industry/antidoping laboratory collaboration to develop and validate screening and confirmation assays so that they would be available before peginesatide reaches the market. We describe a screening ELISA and a confirmation assay consisting of immune-purification followed by separation with SDS-PAGE and revelation with Western double blotting. Both assays can detect 0.5 ng/mL concentrations of peginesatide in blood samples, enabling detection for several days after administration of a physiologically relevant dose. This initial report describes experimental characterization of these assays, including testing with a blinded set of samples from a clinical study conducted in healthy volunteers.

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.

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection.

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4%), followed by genotype 3 (21.4%), and genotype 2 (7.2%). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4%), mild (57.2%), and moderate (21.4%). Viral RNA was detected in liver cells from nine patients (64.3%). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of São Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.

Evaluation of five screening tests licensed in Argentina for detection of hepatitis C virus antibodies

This study was conducted to compare among the most recent generation of five screening tests licensed in Argentina, in order to evaluate which of the tests has the best sensitivity for detection of antibodies against hepatitis C virus (HCV). The tests analyzed were: Detect-HCV™ (3.0) Biochem ImmunoSystems, Canada; Hepatitis C EIA Wiener Lab., Argentina; Equipar HCV Ab, Italy; Murex HCV 4.0, UK and Serodia-HCV particles agglutination test, Japan. The results obtained showed high discrepancy between the different kits used and show that some of the tests assessed have a low sensitivity for anti-HCV detection in both chronic infections and early seroconversion, and indicate that among the commercially available kits in Argentina, Murex HCV 4.0 (UK) and Serodia-HCV particles agglutination test (Japan) have the best sensitivity for HCV screening. Although the sensitivity of the assays is the first parameter to be considered for blood screening, more studies should be carried out to assess the specificity of such assays.

Detection of dengue virus serotype 3 by reverse transcription-polymerase chain reaction in Aedes aegypti (Diptera, Culicidae) captured in Manaus, Amazonas

The detection of dengue virus serotypes from Aedes aegypti in Manaus, state of Amazonas was carried out using the reverse transcription-polymerase chain reaction technique. Fourteen pools out 82 (17.1%) were positive for DENV3, providing a minimal infection rate of 2.1% of all analyzed infected female specimens of three different areas of the city.