Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.

Application of molecular techniques in the study of Staphylococcus aureus clonal evolution - A Review

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.

Association of the presence of residual anti-Toxoplasma gondii IgM in pregnant women and their respective family groups in Miracema, Northwest Rio de Janeiro, Brazil

The seroprevalence of toxoplasmosis in 832 pregnant women in Miracema, Rio de Janeiro, was determined and 75.1% (625) and 2.0% (17) were anti-Toxoplasma gondii IgG and IgM positive, respectively. Out of the 17 IgM positive pregnant women, only one had low avidity IgG corresponding to the acute phase of the infection. All the other women presented with high avidity IgG and also presented with residual IgM anti-T. gondii. Of this sample, 106 received home visits (this includes 11 family nuclei of pregnant women with residual IgM anti-T. gondii, 68 nuclei of only IgG positive pregnant women and 27 nuclei of pregnant women with no antibodies to anti-T. gondii), resulting in 267 individuals visited. Out of these 267 individuals, 21 were positive for IgG and IgM anti-T. gondii and were candidates for the IgG avidity test. All of them presented with high avidity IgG and residual IgM. Five of these IgM+ individuals were (5/238; 2.1%) relatives of IgM negative pregnant women. The other 16 (16/29; 55.2%) were relatives of IgM+ pregnant women who were positive for residual IgM anti-T. gondii. This association was statistically significant (p = 0.0000). The analysis presented herein raises questions regarding the presence of residual IgM anti-T. gondii such as genetic determinants or even constant antigenic stimuli for the same family cluster.

Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species

Aspartyl proteases are a class of enzymes that include the yeast aspartyl proteases and secreted aspartyl protease (Sap) superfamilies. Several Sap superfamily members have been demonstrated or suggested as virulence factors in opportunistic pathogens of the genus Candida. Candida albicans, Candida tropicalis, Candida dubliniensis and Candida parapsilosis harbour 10, four, eight and three SAP genes, respectively. In this work, genome mining and phylogenetic analyses revealed the presence of new members of the Sap superfamily in C. tropicalis (8), Candida guilliermondii (8), C. parapsilosis(11) and Candida lusitaniae (3). A total of 12 Sap families, containing proteins with at least 50% similarity, were discovered in opportunistic, pathogenic Candida spp. In several Sap families, at least two subfamilies or orthologous groups were identified, each defined by > 90% sequence similitude, functional similarity and synteny among its members. No new members of previously described Sap families were found in a Candida spp. clinical strain collection; however, the universality of SAPT gene distribution among C. tropicalis strains was demonstrated. In addition, several features of opportunistic pathogenic Candida species, such as gene duplications and inversions, similitude, synteny, putative transcription factor binding sites and genome traits of SAP gene superfamily were described in a molecular evolutionary context.

Imaging techniques and histology in the evaluation of liver fibrosis in hepatosplenic schistosomiasis mansoni in Brazil: a comparative study

Few publications have compared ultrasound (US) to histology in diagnosing schistosomiasis-induced liver fibrosis (LF); none has used magnetic resonance (MR). The aim of this study was to evaluate schistosomal LF using these three methods. Fourteen patients with hepatosplenic schistosomiasis admitted to hospital for surgical treatment of variceal bleeding were investigated. They were submitted to upper digestive endoscopy, US, MR and wedge liver biopsy. The World Health Organization protocol for US in schistosomiasis was used. Hepatic fibrosis was classified as absent, slight, moderate or intense. Histology and MR confirmed Symmers' fibrosis in all cases. US failed to detect it in one patient. Moderate agreement was found comparing US to MR; poor agreement was found when US or MR were compared to histology. Re-classifying LF as only slight or intense created moderate agreement between imaging techniques and histology. Histomorphometry did not separate slight from intense LF. Two patients with advanced hepatosplenic schistosomiasis presented slight LF. Our data suggest that the presence of the characteristic periportal fibrosis, diagnosed by US, MR or histology, associated with a sign of portal hypertension, defines the severity of the disease. We conclude that imaging techniques are reliable to define the presence of LF but fail in grading its intensity.

Evaluation of rapid techniques for the detection of mycobacteria in sputum with scanty bacilli or clinically evident, smear negative cases of pulmonary and extra-pulmonary tuberculosis

The objective of the current study was to compare two rapid methods, the BBL Mycobacteria Growth Indicator Tube (MGIT TM) and Biotec FASTPlaque TB TM (FPTB) assays, with the conventional Löwenstein-Jensen (LJ) media assay to diagnose mycobacterial infections from paucibacillary clinical specimens. For evaluation of the clinical utility of the BBL MGIT TM and FPTB assays, respiratory tract specimens (n = 208), with scanty bacilli or clinically evident, smear negative cases and non-respiratory tract specimens (n = 119) were analyzed and the performance of each assay was compared with LJ media. MGIT and FPTB demonstrated a greater sensitivity (95.92% and 87.68%), specificity (94.59% and 98.78%), positive predictive value (94.91% and 99.16%) and negative predictive value (96.56% and 90.92%), respectively, compared to LJ culture for both respiratory tract and non-respiratory tract specimens. However, the FPTB assay was unable to detect nontuberculous mycobacteria and few Mycobacterium tuberculosis complex cases from paucibacillary clinical specimens. It is likely that the analytical sensitivity of FPTB is moderately low and may not be useful for the direct detection of tuberculosis in paucibacillary specimens. The current study concluded that MGIT was a dependable, highly efficient system for recovery of M. tuberculosis complexes and nontuberculous mycobacteria from both respiratory and non-respiratory tract specimens in combination with LJ media.

Evaluation of two coproscopic techniques for the diagnosis of schistosomiasis in a low-transmission area in the state of Minas Gerais, Brazil

This population study, which evaluated two parasitological methods for the diagnosis of schistosomiasis mansoni, was performed in a low-transmission area in Pedra Preta, Montes Claros, Minas Gerais, Brazil. A total of 201 inhabitants of the rural area participated in this research. Four stool samples were obtained from all participants and analysed using the Kato-Katz method (18 slides) and a commercial test, the TF-Test®, which was performed quantitatively. The data were analysed to determine prevalence, the sensitivity of the diagnostic methods, the worm burden and the definition of the "gold standard", which was obtained by totalling the results of all samples examined using the Kato-Katz technique and the TF-Test®. The results showed that the prevalence obtained from the examination of one Kato-Katz slide (the methodology adopted by the Brazilian control programme) was 8% compared to 35.8% from the "gold standard", which was a 4.5-fold difference. This result indicates that the prevalence of schistosomiasis in so-called low-transmission areas is significantly underestimated.

The classification of esterases: an important gene family involved in insecticide resistance - A review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and SimplexaTM Dengue real-time RT-PCR confirmed DENV-4 infection in all cases. Additionally, DENV-4 was confirmed in a female Ae. aegypti with 1.08 x 10³ copies/mL of virus, as determined by quantitative real-time RT-PCR. This is the first time the SimplexaTM Dengue real-time assay has been used for the classification of cases of infection and for entomological investigations. The use of these molecular techniques was shown to be important for the surveillance of dengue in humans and vectors.

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g-1)] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g-1, while the threshold for the ST was greater than 0.1.10³ CFU-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.

Comparative serology techniques for the diagnosis of Trypanosoma cruzi infection in a rural population from the state of Querétaro, Mexico

Immunological diagnostic methods for Trypanosoma cruzi depend specifically on the presence of antibodies and parasitological methods lack sensitivity during the chronic and “indeterminate” stages of the disease. This study performed a serological survey of 1,033 subjects from 52 rural communities in 12 of the 18 municipalities in the state of Querétaro, Mexico. We detected anti-T. cruzi antibodies using the following tests: indirect haemagglutination assay (IHA), indirect immunofluorescence assay (IFA), ELISA and recombinant ELISA (rELISA). We also performed Western blot (WB) analysis using iron superoxide dismutase (FeSOD), a detoxifying enzyme excreted by the parasite, as the antigen. Positive test results were distributed as follows: ELISA 8%, rELISA 6.2%, IFA and IHA 5.4% in both cases and FeSOD 8%. A comparative study of the five tests was undertaken. Sensitivity levels, specificity, positive and negative predictive values, concordance percentage and kappa index were considered. Living with animals, trips to other communities, gender, age, type of housing and symptomatology at the time of the survey were statistically analysed using SPSS software v.11.5. Detection of the FeSOD enzyme that was secreted by the parasite and used as an antigenic fraction in WBs showed a 100% correlation with traditional ELISA tests.

Evaluation of parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni in a low transmission area

This study evaluated parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni. A population-based study was performed in 201 inhabitants from a low transmission locality named Pedra Preta, municipality of Montes Claros, state of Minas Gerais, Brazil. Four stool samples were analysed using two techniques, the Kato-Katz® (KK) technique (18 slides) and the TF-Test®, to establish the infection rate. The positivity rate of 18 KK slides of four stool samples was 28.9% (58/201) and the combined parasitological techniques (KK+TF-Test®) produced a 35.8% positivity rate (72/201). Furthermore, a polymerase chain reaction (PCR)-ELISA assay produced a positivity rate of 23.4% (47/201) using the first sample. All 72 patients with positive parasitological exams were treated with a single dose of Praziquantel® and these patients were followed-up 30, 90 and 180 days after treatment to establish the cure rate. Cure rates obtained by the analysis of 12 KK slides were 100%, 100% and 98.4% at 30, 90 and 180 days after treatment, respectively. PCR-ELISA revealed cure rates of 98.5%, 95.5% and 96.5%, respectively. The diagnostic and assessment of cure for schistosomiasis may require an increased number of KK slides or a test with higher sensitivity, such as PCR-ELISA, in situations of very low parasite load, such as after therapeutic interventions.

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1 gene (Nramp1/Slc11a1) is a gene that controls the susceptibility of inbred mice to intracellular pathogens. Polymorphisms in the human Slc11a1/Nramp1 gene have been associated with host susceptibility to leprosy. This study has evaluated nine polymorphisms of the Slc11a1/Nramp1 gene [(GT)n, 274C/T, 469+14G/C, 577-18G/A, 823C/T, 1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19 patients had the multibacillary and the paucibacillary clinical forms of the disease, respectively), and 239 healthy controls matched by age, gender, and ethnicity. The frequency of allele 2 of the (GT)n polymorphism was higher in leprosy patients [p = 0.04, odds ratio (OR) = 1.49], whereas the frequency of allele 3 was higher in the control group (p = 0.03; OR = 0.66). Patients carrying the 274T allele (p = 0.04; OR = 1.49) and TT homozygosis (p = 0.02; OR = 2.46), such as the 469+14C allele (p = 0.03; OR = 1.53) of the 274C/T and 469+14G/C polymorphisms, respectively, were more frequent in the leprosy group. The leprosy and control groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A, 1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the 469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy, while the allele 2 and 3 of the (GT)n polymorphism in the promoter region were associated with susceptibility and protection to leprosy, respectively.