Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the São Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong Nº2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

First identification of natural infection of Rickettsia rickettsii in the Rhipicephalus sanguineus tick, in the State of Rio de Janeiro

The Brazilian Spotted Fever (BSF) is a zoonotic disease caused by Rickettsia rickettsii and transmitted by ticks of the genus Amblyomma, more frequently, Amblyomma cajennense. The aim of this paper was to report the first molecular detection of R. rickettsii on R. sanguineus naturally infected in Rio de Janeiro, Brazil. Ticks were collected from dogs in a rural region of Resende municipality, Rio de Janeiro State, Brazil (22º30'9.46"S, 44º42'44.29"WO), where occurred five human cases of BSF in 2006. The ticks were identified under a stereoscopic microscope and separated in pools by stages, species and sex. DNA extraction was carried out using QIAamp DNA Mini Kit (QIAGEN®). The DNA was submitted to PCR amplification using 04 set of primers: Rr190.70p/Rr190.602n (OmpA, 532bp), BG1-21/BG2-20 (OmpB, 650bp), Tz15/Tz16 (17 kDa protein-encoding gene, 246bp) and RpCS.877p/RpCS.1258n (gltA, 381bp). PCR products were separated by electrophoresis on 1% agarose gels and visualized under ultraviolet light with ethidium bromide. PCR products of the expected sizes were purified by QIAquick® and sequenced by ABI PRISM®. The generated nucleotide sequences were edited with using Bioedit® software and compared with the corresponding homologous sequences available through GenBank, using Discontiguous Mega Blast (http://www.ncbi.nlm.nih.gov). It was confirmed R. rickettsii by sequencing of the material (GenBank FJ356230). The molecular characterization of R. rickettsii in the tick R. sanguineus emphasizes the role of dogs as carriers of ticks from the environment to home. Moreover, this result suggests that there is a considerable chance for active participation of R. sanguineus as one of tick species in the transmission of R. ricketsii to human being in the Brazilian territory.

Difficulties in the diagnosis of HTLV-2 infection in HIV/AIDS patients from Brazil: comparative performances of serologic and molecular assays, and detection of HTLV-2b subtype

The current diagnosis of human T-lymphotropic virus type-2 (HTLV-2) infection is based on the search of specific antibodies; nevertheless, several studies conducted in Brazil pointed deficiencies of the commercially available kits in detecting HTLV-2, mostly in HIV/AIDS patients. This study searched for the presence of HTLV-1 and -2 in 758 HIV/AIDS patients from Londrina, Paraná, Brazil. Serum samples were screened for HTLV-1/2 antibodies using two EIA kits (Vironostika and Murex), and confirmed by WB (HTLV Blot 2.4, Genelabs). The results obtained by EIA disclosed 49 (6.5%) reactive sera: 43 positive by both EIA kits, and six with discordant results. WB confirmed HTLV-1 infection in seven samples (0.9%) and HTLV-2 in 21 sera (2.8%). Negative and indeterminate results were detected in four (0.5%) and 16 (2.1%) sera, respectively. Blood from 47 out of 49 HTLV seroreactive patients were collected and analyzed for the presence of env, LTR and tax genomic segments of HTLVs by PCR. PCR confirmed six cases of HTLV-1 and 37 cases of HTLV-2 infection (14 out of 16 that were found to be WB indeterminate). Restriction analysis of the env PCR products of HTLV-2 disclosed 36 isolates of HTLV-2a/c subtype, and one of HTLV-2b subtype. These results emphasize the need of improving serologic tests for detecting truly HTLV-2 infected patients from Brazil, and confirm the presence of HTLV-2b subtype in the South of this country.

Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

INTRODUCTION: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. METHODS: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5′-3′-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. RESULTS: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95%I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. CONCLUSIONS: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

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.

On line pre-concentration for simultaneous determination of low molecular weight organic acids and inorganic anions in Amazonian river water samples employing ion chromatography with conductivity detection

An ion chromatography procedure, employing an IonPac AC15 concentrator column was used to investigate on line preconcentration for the simultaneous determination of inorganic anions and organic acids in river water. Twelve organic acids and nine inorganic anions were separated without any interference from other compounds and carry-over problems between samples. The injection loop was replaced by a Dionex AC15 concentrator column. The proposed procedure employed an auto-sampler that injected 1.5 ml of sample into a KOH mobile phase, generated by an Eluent Generator, at 1.5 mL min-1, which carried the sample to the chromatographic columns (one guard column, model AG-15, and one analytical column, model AS15, with 250 x 4mm i.d.). The gradient elution concentrations consisted of a 10.0 mmol l-1 KOH solution from 0 to 6.5 min, gradually increased to 45.0 mmol l-1 KOH at 21 min., and immediatelly returned and maintained at the initial concentrations until 24 min. of total run. The compounds were eluted and transported to an electro-conductivity detection cell that was attached to an electrochemical detector. The advantage of using concentrator column was the capability of performing routine simultaneous determinations for ions from 0.01 to 1.0 mg l-1 organic acids (acetate, propionic acid, formic acid, butyric acid, glycolic acid, pyruvate, tartaric acid, phthalic acid, methanesulfonic acid, valeric acid, maleic acid, oxalic acid, chlorate and citric acid) and 0.01 to 5.0 mg l-1 inorganic anions (fluoride, chloride, nitrite, nitrate, bromide, sulfate and phosphate), without extensive sample pretreatment and with an analysis time of only 24 minutes.

Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004

Rotavirus is a major cause of infantile acute diarrhea, causing about 440,000 deaths per year, mainly in developing countries. The World Health Organization has been recommending the assessment of rotavirus burden and strain characterization as part of the strategies of immunization programs against this pathogen. In this context, a prospective study was made on a sample of 134 children with acute diarrhea and severe dehydration admitted to venous fluid therapy in two state hospitals in Rio de Janeiro, Brazil, from February to September 2004. Rotavirus where detected by polyacrylamide gel electrophoresis (PAGE) and by an enzyme-linked immunoassay to rotavirus and adenovirus (EIARA) in 48% of the children. Positive samples for group A rotavirus (n = 65) were analyzed by reverse transcription/heminested multiplex polymerase chain reaction to determine the frequency of G and [P] genotypes and, from these, 64 samples could be typed. The most frequent G genotype was G1 (58%) followed by G9 (40%). One mixed infection (G1/G9) was detected. The only [P] genotype identified was [8]. In order to estimate the rotavirus infection frequency in children who acquired diarrhea as hospital infection in those hospitals, we studied 24 patients, detecting the pathogen in 41% of them. This data suggest that genotype G9 is an important genotype in Rio de Janeiro, with implications to the future strategies of vaccination against rotavirus, reinforcing the need of continuous monitoring of circulating strains of the pathogen, in a surveillance context.

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.

Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus

Human adenoviruses (HAdV) are a major cause of acute respiratory diseases (ARD), gastroenteritis, conjunctivitis and urinary infections. Between November 2000-April 2007, a total of 468 nasopharyngeal aspirate samples were collected from children with ARD at the Clinics Hospital of Uberlândia. These samples were tested by immunofluorescence assay (IFA) and 3% (14/468) tested positive for the presence of HAdV. By performing polymerase chain reaction (PCR) to detect HAdV DNA in samples that tested negative or inconclusive for all viruses identifiable by IFA (respiratory syncytial virus, parainfluenza viruses 1, 2 and 3, influenza viruses A and B and HAdV), as well as negative for rhinoviruses by reverse transcription-PCR, additional 19 cases were detected, for a total of 33 (7.1%) HAdV-positive samples. Nucleotide sequences of 13 HAdV samples were analyzed, revealing that they belonged to species B, C and E. Further analyses showed that species C (HAdV-2) was the most prevalent among the sequenced samples. To our knowledge, this is the first report describing the presence of HAdV-4 in Brazil. We also detected an isolate that was 100% identical to a part of the feline adenovirus hexon gene sequence.

Development of a molecular method for detection and identification of Xanthomonas campestris pv. viticola

In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 10(4) CFU/ml, but this limit could be lowered to 10² CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminaata by in situ hybridization with biotinylated DNA probes

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with bioti-nylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.

Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Serological, epidemiological and molecular aspects of hepatitis C virus infection in a population from Londrina, PR, Brazil, 2001-2002

Serological, epidemiological and molecular aspects of hepatitis C virus (HCV) infection were evaluated in 183 subjects from Londrina, Paraná, Brazil, and adjacent areas. Serum samples which tested anti-HCV positive by microparticle enzyme immunoassay (MEIA) obtained from eight patients with chronic hepatitis C, 48 blood donors, and 127 patients infected with the human immunodeficiency virus (HIV) were submitted to another enzyme immunoassay (ELISA) and to the polymerase chain reaction (PCR). About 78.7% of samples were also reactive by ELISA, with the greater proportion (70.8%) of discordant results verified among blood donors. A similar finding was observed for HCV-RNA detection by PCR, with 111/165 (67.3%) positive samples, with higher rates among HIV-positive subjects and patients with chronic hepatitis than among blood donors. Sixty-one PCR-positive samples were submitted to HCV genotyping, with 77.1, 21.3 and 1.6% of the samples identified as types 1, 3 and 2, respectively. Finally, analysis of some risk factors associated with HCV infection showed that intravenous drug use was the most common risk factor among HIV/HCV co-infected patients, while blood transfusion was the most important risk factor in the group without HIV infection. The present study contributed to the knowledge regarding risk factors associated with HCV infection and the distribution of HCV genotypes in the population evaluated.