Molecular characterization by LSSP-PCR and DNA sequencing of a pathogenic isolate of Leptospira interrogans from Brazil.

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31-21%), Hardjo Norma (46-40%), Hardjo hardjobovis (18-10%), Mini (8-4%) and Wolffi (7-4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three-dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single-specific primer polymerase chain reaction (LSSP-PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis.

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP.

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis.

The pyrazinamidase gene coding for the enzyme that activates the bactericidal drug pyrazinamide contains a polymorphic site that is preserved in Mycobacterium bovis. We synthesized two sets of primers, one encompassing a 180 bp fragment, and the second spanning a 726 bp fragment including the full pncA gene. Following PCR of Mycobacterium tuberculosis and M. bovis samples, it is possible to discriminate by this polymorphism between these species by digestion with Eco065 I. Digestion of the 180 bp fragment results in two fragments of 101 and 79 bp, specific for M. tuberculosis. Alternatively, digestion of the 726 bp fragment yields three fragments of 452, 165 and 109 bp for M. tuberculosis, but only two fragments of 561 and 165 bp for M. bovis.

BCG lymphadenopathy detected in a BCG-vaccinated infant

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73 percent of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96 percent of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

BCG lymphadenopathy detected in a BCG-vaccinated infant.

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73% of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96% of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.