Tuberculosis in a southern Brazilian prison

The occurrence of tuberculosis (TB) in prisons has been described as an alarming public health problem in many countries, especially in developing nations. The objective of this study was to conduct a survey among prisoners with TB respiratory symptoms in order to estimate the incidence of the disease, to analyze the drug susceptibility profile and genotype the isolates of Mycobacterium tuberculosis in the city of Charqueadas, southern of Brazil. The TB incidence was 55/1,900 inhabitants in the prison; this corresponds to an incidence of 3,789/100,000 inhabitants, with a prevalence of 72/1,900 (4,960/100,000 inhabitants). Drug susceptibility test was performed and, among the analyzed isolates, 85% were susceptible to all drugs tested and 15% were resistant to at least one drug, of which 89% were resistant only to isoniazid (INH) or in combination with another drug. The genotype classification of spoligotyping analysis showed that 40% of the isolates belong to LAM family, 22% to T family, 17.5% to Haarlem family, 12.5% to U family and 3% to X family. The shared international spoligotypes most frequently found were 729 (27%), 50 (9.5%), 42 (8%), 53 (8%) and 863 (8%). In conclusion, it was observed that TB in this specific population had been caused, mostly, by strains that have been transmitted in the last few years, as demonstrated by the large level of genotype clustering. In addition, it was found specific large clusters, which were not often found in the general population from the same period and in the same region.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3 percent and 98.1 percent, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6 percent and 100 percent, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100 percent consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Internal control in PCR for Mycobacterium tuberculosis: usefulness and improvement of the diagnosis

The aim of this work was to construct and test a plasmidial Internal Control (IC) to detect the inhibition in the PCR test for M. tuberculosis and also its contribution for a Public Health Laboratory routine. The IC was a 600-bp of DNA linked to a plasmid with the same primer sites, allowing the amplification with the 245-bp diagnostic fragment. The amplification of the positive samples rendered the IC and the diagnostic fragment; instead negative samples only showed the IC. A total of 149 tuberculosis samples were studied and introduced the IC to monitor. Results showed 3.3% of the samples without amplification of the IC, suggesting the inhibition. These samples showed results in accordance with the clinical results. The objective of the IC was to identify the false negative results.

A PCR do elemento IS6110 para diagnóstico da Tuberculose (TB) é muito utilizada em laboratórios de pesquisa. As suas limitações incluem, a inibição da enzima Taq DNA Polimerase. A seguir descrevemos a construção de um Controle Interno (IC) e ensaios de detecção da inibição da PCR para M. tuberculosis. O IC é um fragmento de DNA de 600 pb com os mesmos sítios de anelamento do primer, permitindo a amplificação com o fragmento diagnóstico de 245 pb. As amostras positivas fornecem um padrão de bandas referentes ao IC (664 pb) e ao fragmento diagnóstico (245 pb), e as amostras negativas apresentam apenas o fragmento correspondente ao IC. 149 amostras com diagnóstico conhecido foram analisadas por PCR introduzindo o IC em todas elas. Os resultados mostraram 3.3% de amostras sem amplificação do IC sugerindo inibição. Estas amostras quando testadas novamente mostraram resultados concordantes com os resultados clínicos. O objetivo do IC e identificar os falsos resultados negativos.

In search of essentiality: Mollicute-specific genes shared by twelve genomes

Mollicutes are cell wall-less bacteria with a genome characterized by its small size. Chromosomal rearrangements help these organisms evade host immune surveillance and hence cause disease. Our goal was to determine genes shared by Mollicutes genomes using the bidirectional best hit methodology. The twelve studied Mollicutes share 210 genes, most of which (> 60 percent) fall into the following COG categories: translation, ribosomal structure and biogenesis; DNA replication, recombination and repair; nucleotide transport and metabolism and energy production and conversion. Thirty Mollicute-specific genes were identified, 22 of them previously described as essential genes in Mycoplasma genitalium.

Genes involved in cell division in mycoplasmas

Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw) cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

Restriction-modification systems in Mycoplasma spp

Restriction and Modification (R-M) systems are present in all Mycoplasma species sequenced so far. The presence of these genes poses barriers to gene transfer and could protect the cell against phage infections. The number and types of R-M genes between different Mycoplasma species are variable, which is characteristic of a polymorphism. The majority of the CDSs code for Type III R-M systems and particularly for methyltransferase enzymes, which suggests that functions other than the protection against the invasion of heterologous DNA may exist. A possible function of these enzymes could be the protection against the invasion of other but similar R-M systems. In Mycoplasma hyopneumoniae strain J, three of the putative methyltransferase genes were clustered in a region forming a genomic island. Many R-M CDSs were mapped in the vicinity of transposable elements suggesting an association between these genes and reinforcing the idea of R-M systems as mobile selfish DNA. Also, many R-M genes present repeats within their coding sequences, indicating that their expression is under the control of phase variation mechanisms. Altogether, these data suggest that R-M systems are a remarkable characteristic of Mycoplasma species and are probably involved in the adaptation of these bacteria to different environmental conditions.

Diagnosis of abdominal angiostrongyliasis by PCR from sera of patients

Angiostrongilíase abdominal é uma infecção zoonótica causada por um parasito nematódeo intravascular de roedores silvestres, Angiostrongylus costaricensis. Nenhum diagnóstico parasitológico é atualmente disponível e o imunodiagnóstico apresenta alguns obstáculos. Oligonucleotídeos foram construídos baseados em um gênero específico, Angiostrongylus cantonensis, e foi capaz de amplificar um fragmento de 232 bp de amostras de soro de 3 pacientes com diagnóstico histopatológico. O melhor método de extração foi com DNAzol e a especificidade dos oligonucleotídeos foi confirmada por Southern Blot. A doença tem sido diagnosticada com freqüência no sul do Brasil, assim, este método surge como uma importante e inédita alternativa no auxílio do diagnóstico desta doença.

Echinococcus granulosus recombinant antigens

Echinococcus granulosus is a small parasitic platyhelminth, the larval stage of which is the causative agent of cystic hydatid disease both in man and domestic ungulates. This zoonosis constitutes major economic and public (both human and veterinary) health problems in many parts of the world, including southern Brazil, where it is hyperendemic. Serodiagnosis of cystic hydatid disease has been of great importance in clinical diagnosis, posttreatment surveillance of patients and epidemiólogical surveys, since it is the most specific of the noninvasive alternatives for detecting infections with the E. granulosus metacestode. Nevertheless, immunological tests present chronical problems associated with the quality and availability of parasite antigens. In this sense, the cloning of E. granulosus antigen-encoding genes comes forth as a valuable alternative for the production of pure and well characterized antigens. In the last few years, several research groups have cloned and characterized genes that encode E. granulosus antigens, many of which are potentially useful in the immunodiagnosis of cystic hydatid disease. Furthermore, several of these antigens represent important components of the parasite biology and may be particularly relevant to vaccination, immunotherapy, or as potential targets for chemotherapy. In this review we summarize the available data concerning the production and characterization of E. granulosus recombinant antigens. We also discuss some of the perspectives opened by the use of molecular biology techniques in the diagnosis of cystic hydatid disease as well as in the study of the biology of this parasite.