St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

New data on Echinococcus spp. in Southern Brazil

40 Echinococcus isolates from sheep and cattle in Southern Brazil were genetically analysed in order to obtain further data on the presence of different taxa of the Echinococcus granulosus complex. Differentiation was done using a PCR technique and sequencing of mitochondrial cytochrome c oxidase subunit 1 (CO1). Most samples (38) could be allocated to the sheep strain (G1) of E. granulosus, while two samples belonged to E. ortleppi, previously known as cattle strain (G5) of E. granulosus. Due to the shorter prepatent period in dogs of the latter taxon, this records have important implications for the design of control measures in this endemic region.

PCR-based identification of Burkholderia pseudomallei

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Occurrence of Proteus mirabilis associated with two species of venezuelan oysters

The fecal contamination of raw seafood by indicators and opportunistic pathogenic microorganisms represents a public health concern. The objective of this study was to investigate the presence of enteric bacteria colonizing oysters collected from a Venezuelan touristic area. Oyster samples were collected at the northwestern coast of Venezuela and local salinity, pH, temperature, and dissolved oxygen of seawater were recorded. Total and fecal coliforms were measured for the assessment of the microbiological quality of water and oysters, using the Multiple Tube Fermentation technique. Analyses were made using cultures and 16S rRNA gene sequencing. Diverse enrichment and selective culture methods were used to isolate enteric bacteria. We obtained pure cultures of Gram-negative straight rods with fimbriae from Isognomon alatus and Crassostrea rhizophorae. Our results show that P. mirabilis was predominant under our culture conditions. We confirmed the identity of the cultures by biochemical tests, 16S rRNA gene sequencing, and data analysis. Other enterobacteria such as Escherichia coli, Morganella morganii and Klebsiella pneumoniae were also isolated from seawater and oysters. The presence of pathogenic bacteria in oysters could have serious epidemiological implications and a potential human health risk associated with consumption of raw seafood.

Predictive factors of mortality in burn patients

Burn mortality statistics may be misleading unless they account properly for the many factors that can influence outcome. Such estimates are useful for patients and others making medical and financial decisions concerning their care. This study aimed to define the clinical, microbiological and laboratorial predictors of mortality with a view to focus on better burn care. Data were collected using independent variables, which were analyzed sequentially and cumulatively, employing univariate statistics and a pooled, cross-sectional, multivariate logistic regression to establish which variables better predict the probability of mortality. Survivors and non-survivors among burn patients were compared to define the predictive factors of mortality. Mortality rate was 5.0%. Higher age, larger burn area, presence of fungi in the wound, shorter length of stay and the presence of multi-resistant bacteria in the wound significantly predicted increased mortality. The authors conclude that those patients who are most apt to die are those with age > 50 years, with limited skin donor sites and those with multi-resistant bacteria and fungi in the wound.

Corynebacterium striatum infecting a malignant cutaneous lesion: the emergence of an opportunistic pathogen

We described a case of a 27-year old male patient with skin and soft tissue infection of a neoplastic lesion caused by Corynebacterium striatum, an organism which has been rarely described as a human pathogen. Identification was confirmed by DNA sequencing. Successful treatment with penicillin was achieved. The role of the C. striatum as an emerging opportunistic pathogen is discussed.

High prevalence and association of HIV-1 non-B subtype with specific sexual transmission risk among antiretroviral naïve patients in Porto Alegre, RS, Brazil

In South Brazil the circulation of two HIV-1 subtypes with different characteristics represents an important scenario for the study of the impact of HIV-1 diversity on the evolution of the HIV-1 epidemic and AIDS disease. HIV-1 B, the predominant variant in industrialized countries and HIV-1 C, the most prevalent subtype in areas with rapid epidemic growth, are implicated in most infections. We evaluated blood samples from 128 antiretroviral (ARV) naïve patients recruited at entry to the largest HIV outpatient service in Porto Alegre. Based on partial pol region sequencing, HIV-1 C was observed in 29%, HIV-1 B in 22.6% and, the recently identified CRF31_BC, in 23.4% of 128 volunteers. Other variants were HIV-1 F in 10% and other mosaics in 5.5%. In order to evaluate the association of socio-behavioral characteristics and HIV-1 subtypes, interviews and laboratory evaluation were performed at entry. Our data suggest an established epidemic of the three major variants, without any evidence of partitioning in either of the subgroups analyzed. However, anal sex practices were associated with subtype B, which could indicate a greater transmissibility of non-B variants by vaginal intercourse. This study provides baseline information for epidemiologic surveillance of the changes of the molecular characteristics of HIV-1 epidemics in this region.

Distribution of hepatitis B virus genotypes and viral load levels in Brazilian chronically infected patients in São Paulo city

The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68% samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2%) and 3 (9.8%). Subtype 1a (82.3%) was the most prevalent, followed by subtypes 3a (9.8%), 1b (5.9%) and 1a/1b (2.0%). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5%) as genotype 1, subtypes 1a, and 8 (9.5%) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100% and 95.2% of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.

Antemortem diagnosis of human rabies in a veterinarian infected when handling a herbivore in Minas Gerais, Brazil

The Ministry of Health's National Human Rabies Control Program advocates pre-exposure prophylaxis (PEP) for professionals involved with animals that are at risk of contracting rabies. We report an antemortem and postmortem diagnosis of rabies in a veterinarian who became infected when handling herbivores with rabies. The antemortem diagnosis was carried out with a saliva sample and a biopsy of hair follicles using molecular biology techniques, while the postmortem diagnosis used a brain sample and conventional techniques. The veterinarian had collected samples to diagnose rabies in suspect herbivores (bovines and caprines) that were subsequently confirmed to be positive in laboratory tests. After onset of classic rabies symptoms, saliva and hair follicles were collected and used for antemortem diagnostic tests and found to be positive by RT-PCR. Genetic sequencing showed that the infection was caused by variant 3 (Desmodus rotundus), a finding confirmed by tests on the brain sample. It is essential that professionals who are at risk of infection by the rabies virus undergo pre-exposure prophylaxis. This study also confirms that molecular biology techniques were used successfully for antemortem diagnosis and therefore not only allow therapeutic methods to be developed, but also enable the source of infection in human rabies cases to be identified accurately and quickly.

Dermatophytoses in domesticated animals

Dermatophytes are among the most frequent causes of ringworm infections in domesticated animals. They are known to serve as reservoirs of the zoophilic dermatophytes and these infections have important zoonotic implication. In Nigeria and probably West Africa, there are not many studies on the incidence of dermatophytosis in domesticated animals. In the current study, 538 domesticated animals with clinically suggestive lesions were investigated for dermatophytes. Identification of dermatophyte species was performed by macro- and micro morphological examination of colonies and by biochemical methods. In the cases of isolates that had atypical morphology and/or biochemical test results, the rDNA internal transcribed spacer region 2 (ITS 2) sequencing was performed. Out of this number, 214 (39.8%) were found to be colonized by a variety of ten species of dermatophytes. M. canis was the most frequently isolated species (37.4%), followed by T. mentagrophytes (22.9%) and T. verrucosum (15.9%). M. persicolor and T. gallinae were jointly the least species isolated with a frequency of 0.55% respectively. The recovery of dermatophyte isolates previously shown to be common etiological agents of dermatophytosis especially from children in the same region suggests that animal to human transmission may be common. Possible implications and recommendations are discussed.

Amplification of the flgE gene provides evidence for the existence of a Brazilian borreliosis

INTRODUCTION: The symptoms of Brazilian borreliosis resemble the clinical manifestations of Lyme disease (LD). However, there are differences between the two in terms of epidemiological and laboratory findings. Primers usually employed to diagnose LD have failed to detect Borrelia strains in Brazil. OBJECTIVE: We aimed to identify the Brazilian Borrelia using a conserved gene that synthesizes the flagellar hook (flgE) of Borrelia burgdorferi sensu lato. METHOD: Three patients presenting with erythema migrans and positive epidemiological histories were recruited for the study. Blood samples were collected, and the DNA was extracted by commercial kits. RESULTS: The gene flgE was amplified from DNA of all selected patients. Upon sequencing, these positive samples revealed 99% homology to B. burgdorferi flgE. CONCLUSION: These results support the existence of borreliosis in Brazil. However, it is unclear whether this borreliosis is caused by a genetically modified B. burgdorferi sensu stricto or by a new species of Borrelia spp.

Genetic diversity and primary resistance among HIV-1-positive patients from Maringá, Paraná, Brazil

The objective of this study is to identify subtypes of Human Immunodeficiency Virus type 1 (HIV-1) and to analyze the presence of mutations associated to antiretroviral resistance in the protease (PR) and reverse transcriptase (RT) regions from 48 HIV-1 positive treatment naïve patients from an outpatient clinic in Maringá, Paraná, Brazil. Sequencing was conducted using PR, partial RT and group-specific antigen gene (gag) nested PCR products from retrotranscribed RNA. Transmitted resistance was determined according to the Surveillance Drug Resistance Mutation List (SDRM) algorithm. Phylogenetic and SimPlot analysis of concatenated genetic segments classified sequences as subtype B 19/48 (39.6%), subtype C 12/48 (25%), subtype F 4/48 (8.3%), with 13/48 (27.1%) recombinant forms. Most recombinant forms were B mosaics (B/F 12.5%, B/C 10.4%), with one C/F (2.1%) and one complex B/C/F mosaic (2.1%). Low levels of transmitted resistance were found in this study, 2/48 (2.1% to NRTIs and 2.1% for PI). This preliminary data may subsidize the monitoring of the HIV evolution in the region.

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.