Alexandre Ribeiro Bello

Identification of Giardia lamblia Assemblage E in Humans Points to a New Anthropozoonotic Cycle

Giardia lamblia is a pathogen transmitted by water and food that causes infection worldwide. Giardia genotypes are classified into 8 assemblages (A-H). Assemblages A and B are detected in humans, but they are potentially zoonotic because they infect other mammalian hosts. Giardia in samples from 44 children was genotyped. Conserved fragments of the genes encoding β-giardin and glutamate dehydrogenase were sequenced and their alignment were carried out with sequences deposited in GenBank. As expected for Rio de Janeiro, the majority of samples were related to assemblage A. Surprisingly, assemblage E was detected in 15 samples. Detection of assemblage E in humans suggests a new zoonotic route of Giardia transmission.

Detection of Toxoplasma gondii DNA by Polymerase Chain Reaction in Experimentally Desiccated Tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

β-lactamase producing enterobacteria isolated from surveillance swabs of patients in a Cardiac Intensive Care Unit in Rio de Janeiro, Brazil

There is a high incidence of infections caused by betalactamase-producing Gram-negative microorganisms in Brazil. These organisms are of clinical and epidemiological importance, since their mobile genetic elements facilitate cross-infection. The present study was conducted in sentinel rectal swabs from patients admitted to a cardiac surgery hospital in Rio de Janeiro, from January through December 2007, in a consecutive manner. The aim of the study was to characterize the genotype and phenotype of these isolates from colonized patients. Biochemical tests, antimicrobial susceptibility tests, a confirmatory test for the expression of extended spectrum betalactamase (ESBL) production and polymerase chain reaction for the blaTEM, blaSHV, CTX-M1, Toho-1 and AmpC genes were performed at the University Hospital of Universidade do Estado do Rio de Janeiro (UERJ). The most frequently isolated bacteria were Escherichia coli 9/41 (21.95%) and Klebsiella pneumoniae 14/41 (34.1%). In 24/41 (58%), the ESBL genotype was confirmed. The most prevalent genes in samples that expressed ESBL were blaTEM 13/24 (54%), AmpC 12/24 (50%), blaSHV 6/24 (25%), CTX-M1 7/24 (29%), and Toho-1 6/24 (25%). Of these, 14/24 (58%) presented more than one genotype for the tested primers. In nine (37%) samples other than E. coli, K. pneumoniae or Proteus spp., the phenotype for ESBL was found and confirmed by PCR. The most sensitive substrate in the approximation test in ESBL positive samples was ceftriaxone (83%). Fifty percent of the samples expressed AmpC were associated with other genes. Intermediate susceptibility to ertapenem was found in 2/41 (5%).

Ocorrência de Cryptosporidium sp em amostras fecais de crianças, menores de 10 anos de idade, com indicação clínica de Rotavírus

The presence of oocysts of Cryptosporidium sp was investigated in 485 fecal samples of children with clinical indication of Rotavirus. No significant differences were observed between Cryptosporidium sp. and rotavirus occurrence and fecal consistency. Cryptosporidium sp also should be performed in the laboratory diagnosis of diarrheic episodes in children.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenotypical gentamicin resistance amplification (frequencies of 10(-3) to 10(-5), compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromosomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

The presence of Giardia lamblia assemblage A in dogs suggests an anthropozoonotic cycle of the parasite in Rio de Janeiro, Brazil

BACKGROUND Giardia lamblia is a zoonotic protozoan that is classified into 8 genotypes and is distributed worldwide. Assemblages A and B were found to infect dogs and humans, whereas assemblages C and D are dog host-specific. Our objective was to investigate the G. lamblia genotypes circulating in a canine population in Rio de Janeiro, RJ. RESULTS Sixty stool samples positive for G. lamblia from street dogs were characterized. Fragments of the conserved genes encoding beta-giardin (β-gia) and glutamate dehydrogenase (gdh) were used as targets. The sequences from beta-giardin and glutamate dehydrogenase genes obtained from all 60 dog samples were 100% similar to G. lamblia genotype A. CONCLUSION The detection of genotype A suggests that G. lamblia transmission in Rio de Janeiro has a predominantly anthropozoonotic cycle.