Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Efficacy of benznidazol treatment for asymptomatic chagasic patients from state of Rio Grande do Sul evaluated during a three years follow-up

The efficacy of benznidazol on the treatment of chagasic patients from the state of Rio Grande do Sul was evaluated during a three-year follow-up. A cohort of 80 asymptomatic chronic chagasic patients or blood bank donors (49 male and 31 female) was studied. Their ages varied from 17-42 years, with a mean and a median of 30 and 35 years, respectively. The 80 patients presented positive serology, hemoculture and polymerase chain reaction (PCR). They were treated with 5 mg/Kg benznidazol twice a day for 60 days. Serological, parasitological and PCR methods were used to evaluate response. Serology was performed using commercial ELISA and indirect immunofluorescence (IFI) tests, parasitemia was monitored by hemoculture in LIT medium and PCR with primers S35/S36 was used to amplify a Trypanosoma cruzi 330 bp kDNA repetitive sequence. PCR positivity of 240 seropositive individuals was compared using DNA preparations from whole blood/guanidine EDTA (GE), buffy-coat/GE and frozen buffy-coat. Fifty non-chagasic individuals were used as negative controls. PCR positivity was 86.7% for the frozen buffy-coat, 71.7% for the GE/buffy-coat and 69.2% for the GE/whole blood. The hemocultures became negative just after treatment and remained negative during the three years of follow-up. In the third year after treatment, 9/80 (11.3%) patients presented negative PCR and, from those, four also presented negative serological tests. Furthermore, a reduction in three serological titers was observed in 27/80 (33.8%) of the patients treated. Taken together, the results show that four of the 80 (5.0%) chronic chagasic patients from the state of Rio Grande do Sul were cured after treatment with benznidazol.

Molecular characterisation of Sporothrix schenckii isolates from humans and cats involved in the sporotrichosis epidemic in Rio de Janeiro, Brazil

An epidemic of sporotrichosis, a subcutaneous mycosis caused by the fungus Sporothrix schenckii, is ongoing in Rio de Janeiro, Brazil, in which cases of human infection are related to exposure to cats. In an attempt to demonstrate the zoonotic character of this epidemic using molecular methodology, we characterised by DNA-based typing methods 19 human and 25 animal S. schenckii isolates from the epidemic, as well as two control strains. To analyse the isolates, the random amplified polymorphic DNA (RAPD) technique was performed using three different primers, together with DNA fingerprinting using the minisatellite derived from the wild-type phage M13 core-sequence. The analyses generated amplicons with considerable polymorphism. Although isolates exhibited high levels of genetic relatedness, they could be clustered into 5-10 genotypes. The RAPD profiles of epidemic S. schenckii isolates could be distinguished from that of the United States isolate, displaying 20% similarity to each primer and 60% when amplified with the M13 primer. DNA fingerprinting of S. schenckii isolated from the nails (42.8%) and the oral cavities (66%) of cats were identical to related human samples, suggesting that there is a common infection source for animals and humans in this epidemic. It is clear that cats act as a vehicle for dissemination of S. schenckii.

Simultaneous infection of human host with genetically distinct isolates of Paracoccidioides brasiliensis

This study is the first report on genetic differences between isolates of Paracoccidioides brasiliensis from a single patient. We describe a simultaneous infection with genetically distinct isolates of P. brasiliensis in a patient with chronic paracoccidioidomycosis. The clinical isolates were obtained from lesions in different anatomical sites and were characterised by random amplified polymorphic DNA (RAPD) analysis. The RAPD technique can be helpful for distinguishing between clinical isolates. Different random primers were used to characterise these clinical isolates. The RAPD patterns allowed for differentiation between isolates and the construction of a phenetic tree, which showed more than 28% genetic variability in this fungal species, opening new possibilities for clinical studies of P. brasiliensis. Based on these results and preliminary clinical findings, we suggest that different genotypes of P. brasiliensis might infect the same patient, inducing the active form of the disease.

Sequencing of E2 and NS5A regions of HCV genotype 3a in Brazilian patients with chronic hepatitis

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Helicobacter pylori cagA and vacA genotypes in Cuban and Venezuelan populations

The aim of this study was to determine the presence of Helicobacter pylori cytotoxin-associated gene (cagA)/vacuolating cytotoxin gene (vacA) among patients with chronic gastritis in Cuba and Venezuela. Gastric antrum biopsies were taken for culture, DNA extraction and PCR analysis. Amplification of vacA and cagA segments was performed using two regions of cagA: 349 bp were amplified with the F1/B1 primers and the remaining 335 bp were amplified with the B7629/B7628 primers. The VA1-F/VA1-R set of primers was used to amplify the 259-bp (s1) or 286-bp (s2) product and the VAG-R/VAG-F set of primers was used to amplify the 567-bp (m1) or 642-bp (m2) regions of vacA. cagA was detected in 87% of the antral samples from Cuban patients and 80.3% of those from Venezuelan patients. All possible combinations of vacA regions were found, with the exception of s2/m1. The predominant combination found in both countries was s1/m1. The percentage of cagA+ strains was increased by the use of a second set of primers and a greater number of strains was amplified with the B7629/B7628 primers in the Cuban patients (p = 0.0001). There was no significant difference between the presence of the allelic variants of vacA and cagA in both populations. The predominant genotype was cagA+/s1m1 in both countries. The results support the necessary investigation of isolates circulating among the human population in each region.

Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Occurrence of blaOXA-23 gene in imipenem-susceptible Acinetobacter baumannii

The aim of the current study was to describe the occurrence of the blaOXA-23 gene and the ISAba1 element in imipenem-susceptible Acinetobacter baumannii strains. By performing the polymerase chain reaction mapping using combinations of ISAba1 forward primers and the blaOXA-23-like gene reverse primers, we demonstrated that the ISAba1 element did not occur upstream of the blaOXA-23 gene in five of 31 isolates, which explained the lack of resistance to imipenem despite the presence of the blaOXA-23 gene. All of the blaOXA-23-positive isolates were susceptible to imipenem and meropenem with minimal inhibitory concentration < 4 µg/mL. Pulsed-field gel electrophoresis analysis revealed four genotypes among the five blaOXA-23-positive isolates. The current report of the blaOXA-23 gene in imipenem-susceptible isolates provided evidence that this gene may be silently spread in a hospital environment and highlighted the threat of undetected reservoirs of carbapenemase genes.

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.

A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures

Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV), mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

Human bocavirus 1 and 3 infection in children with acute gastroenteritis in Brazil

To determine the positivity rate of human bocavirus (HBoV) 1 and 3 among children who presented with acute gastroenteritis symptoms during the period of 1994-2004 in the Central-West Region of Brazil, 762 faecal samples were tested using polymerase chain reaction (PCR) for the detection of HBoV DNA. Primers for a segment of the non-structural viral protein 1 (NS1) gene of HBoV-1 and HBoV-3 were used. Twelve HBoV-positive samples were further characterised via genomic sequencing and phylogenetic analysis. Of the samples tested, 5.8% (n = 44) were positive for HBoV-1 or HBoV-3 and co-infection was observed in 14 (31.8%) of the 44 HBoV-positive samples. Nine of the 14 samples were also positive for Rotavirus A and five were positive for Aichi virus. The genomic sequencing of the NS1 partial sequence of 12 HBoV-samples showed that 11 samples were characterised as HBoV-1 and that one was characterised as HBoV-3. The phylogenetic analysis showed that the HBoV-1 samples had a high sequence homology to others previously identified in China, Sweden and Brazil. This is the first study conducted in the Central-West Region of Brazil to detect HBoV-1 and HBoV-3 in faecal samples from children with acute gastroenteritis. Further studies are required to define the role of HBoVs as aetiological agents of gastroenteritis.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.