Molecular characterisation of multidrug-resistant Mycobacterium tuberculosis isolates from a high-burden tuberculosis state in Brazil.

Tuberculosis (TB) is the leading cause of death among infectious diseases worldwide. Among the estimated cases of drug-resistant TB, approximately 60% occur in the BRICS countries (Brazil, Russia, India, China and South Africa). Among Brazilian states, primary and acquired multidrug-resistant TB (MDR-TB) rates were the highest in Rio Grande do Sul (RS). This study aimed to perform molecular characterisation of MDR-TB in the State of RS, a high-burden Brazilian state. We performed molecular characterisation of MDR-TB cases in RS, defined by drug susceptibility testing, using 131 Mycobacterium tuberculosis (M.tb) DNA samples from the Central Laboratory. We carried out MIRU-VNTR 24loci, spoligotyping, sequencing of the katG, inhA and rpoB genes and RDRio sublineage identification. The most frequent families found were LAM (65.6%) and Haarlem (22.1%). RDRio deletion was observed in 42 (32%) of the M.tb isolates. Among MDR-TB cases, eight (6.1%) did not present mutations in the studied genes. In 116 (88.5%) M.tb isolates, we found mutations associated with rifampicin (RIF) resistance in rpoB gene, and in 112 isolates (85.5%), we observed mutations related to isoniazid resistance in katG and inhA genes. An insertion of 12 nucleotides (CCAGAACAACCC) at the 516 codon in the rpoB gene, possibly responsible for a decreased interaction of RIF and RNA polymerase, was found in 19/131 of the isolates, belonging mostly to LAM and Haarlem families. These results enable a better understanding of the dynamics of transmission and evolution of MDR-TB in the region.

Association of slow N-acetyltransferase 2 profile and anti-TB drug-induced hepatotoxicity in patients from Southern Brazil.

PURPOSE: To determine the frequency of N-acetyltransferase 2 (NAT2) polymorphisms, the NAT2 acetylation profile and its relation to the incidence of gastrointestinal adverse drug reactions (ADRs), anti-tuberculosis (TB) drug-induced hepatotoxicity, and the clinical risk factors for hepatotoxicity in a population from Brazil. METHODS: Two hundred and fifty-four Brazilian TB patients using isoniazid (INH), rifampicin (RMP), and pirazinamide (PZA) were tested in a prospective cohort study. NAT2 genotyping was performed by direct PCR sequencing. The association between gastrointestinal ADRs/hepatotoxicity and the NAT2 profile genotype was evaluated by univariate analysis and multiple logistic regression. RESULTS: Of the 254 patients analyzed, 69 (27.2%) were slow acetylators and 185 (72.8%) were fast acetylators. Sixty-five (25.6%) patients were human immunodeficiency virus (HIV)-positive. Thirty-three (13%) and 14 (5.5%) patients developed gastrointestinal ADR and hepatotoxicity, respectively. Of the 14 hepatotoxicity patients, nine (64.3%) were slow acetylators and five (35.7%) were fast acetylators. Sex, age, presence of hepatitis C virus, alcohol abuse, and baseline aminotransferases were not found to be risk factors for hepatotoxicity. However, logistic regression analysis revealed that slow acetylator status and the presence of HIV (p < 0.05) were independent risk factors for hepatotoxicity. CONCLUSIONS: Our findings show that HIV-positive patients that have the slow acetylation profile are significantly associated with a higher risk of developing hepatotoxicity due to anti-TB drugs.

Epidemiology of meningococcal disease in southern Brazil from 1995 to 2003, and molecular characterization of Neisseria meningitidis using multilocus sequence typing.

OBJECTIVE: To describe the epidemiology of meningococcal disease (MD) in southern Brazil. METHODS: Retrospective cohort study among 2215 MD cases reported from 1995 to 2003 in Rio Grande do Sul (RS) State. RESULTS: The overall incidence fell by 50%; the case-fatality rate during this period was 22%. Even so, the incidence of MD remained high after the epidemic period ended in 1999. Together, the age groups of 1-4 years and infants accounted for 54.1% of reported cases with incidences of 11.3/100 000 and 31.3/100 000, respectively; 69.8% of cases were caused by Neisseria meningitidis serogroup B, which increased significantly. There was a significant decrease in serogroup C cases in the whole period. The phenotypes B:4,7:P1.19,15, B:15:P1.7,16 and B:NT:P1.3 caused almost 50% of all serotyped cases. Fifty-six isolates obtained from RS patients during the first non-epidemic year 2000 plus 20 isolates from other southern Brazilian states (Santa Catarina and Paraná), Denmark and France were typed by multilocus sequence typing. Twenty sequence types (STs) were identified, eight of them found only in RS. ST-33 (27%) and ST-259 (18%) were the most frequent; both belong to the ST-32/ET-5 complex. ST-259 cases showed a trend towards higher risk of fatal outcome. ST-259 isolates were not detected among geographic controls or in other studies in Brazil. CONCLUSION: Our data suggest that ST-33 and ST-259 clones and the emergence of the ST-103 isolates contributed to the continued high incidence of MD in RS.

Recent transmission of tuberculosis involving retired patients.

The reported incidence of tuberculosis (TB) in three different regions of Rio Grande do Sul State in Brazil varies considerably. We used IS6110-RFLP and spoligotyping methods to genotype Mycobacterium tuberculosis isolates obtained from 268 patients between 1998 and 2000 in order to assess the levels of recent transmission of TB in the three regions. The degree of clustering of the strain types did not differ among the three regions; neither did other characteristics such as demographic features, underlying medical conditions, or the proportion of resistant TB. As reported previously, male patients were at greater risk of developing TB and our data suggest that part of this may be related to the higher rates of recent transmission among them (P<0.05). In addition, we found that retired patients were almost 3 times more likely to be infected with cluster-pattern strains than patients reporting any other occupation (P<0.05), and more than 3 times more likely than non-retired patients in the same age group (P<0.05) to be infected with cluster-pattern strains. We conclude that recent transmission is not a major factor contributing to the differences in TB incidence in the three regions of Rio Grande do Sul. The reason for the suggested high proportion of recent transmission TB cases among the retired people needs further studies.

Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.

SETTING: A public health laboratory in a tuberculosis-endemic region in Brazil. OBJECTIVE: To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory. DESIGN: Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe. RESULTS: Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol. CONCLUSION: The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory.

A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2x10(2) c.f.u. ml(-1). Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.

Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli

1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro