Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis.

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.

Detection of Mycobacterium bovis DNA in nasal swabs from tuberculous cattle by a multiplex PCR.

Detection of tuberculosis in cattle relies on the intradermal tuberculin test (ITT), but a definitive diagnosis requires identification of the pathogen after the animal is slaughtered. DNA in nasal swabs from 50 cows was analyzed by m-PCR, targeting for the RvD1-Rv2031c and IS6110 sequences. M. bovis was identified in two of 34 tuberculous cows (5.9%). The use of mPCR of nasal swabs as an in vivo diagnostic tool for bovine tuberculosis is suggested.

Epidemic of postsurgical infections caused by Mycobacterium massiliense.

An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most (n = 144; 97.2%) isolates presented a PRA-hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense. Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense. Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC(90)], 8 microg/ml) and clarithromycin (MIC(90), 0.25 microg/ml) but resistance to ciprofloxacin (MIC(90), >or=32 microg/ml), cefoxitin (MIC(90), 128 microg/ml), and doxycycline (MIC(90), >or=64 microg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.

Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.

BACKGROUND: Mutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains. RESULTS: We, therefore, conducted a comprehensive evaluation of INH resistant M. tuberculosis strains (n = 224) from three South American countries with high burden of drug resistant TB to characterize mutations in katG, ahpC and inhA gene loci and correlate with minimal inhibitory concentrations (MIC) levels and spoligotype strain family. Mutations in katG were observed in 181 (80.8%) of the isolates of which 178 (98.3%) was contributed by the katG S315T mutation. Additional mutations seen included oxyR-ahpC; inhA regulatory region and inhA structural gene. The S315T katG mutation was significantly more likely to be associated with MIC for INH >or=2 microg/mL. The S315T katG mutation was also more frequent in Haarlem family strains than LAM (n = 81) and T strain families. CONCLUSION: Our data suggests that genetic screening for the S315T katG mutation may provide rapid information for anti-TB regimen selection, epidemiological monitoring of INH resistance and, possibly, to track transmission of INH resistant strains.

Influence of potentially remineralizing agents on bleached enamel microhardness.

This study investigated the effect of the addition of calcium and fluoride into a 35% hydrogen peroxide gel on enamel surface and subsurface microhardness. Twenty extracted human third molars were sectioned to obtain enamel fragments and they were divided into four groups (n = 20) according to the bleaching treatment. Group 1 received no bleaching procedure (control). Group 2 was treated with a 35% hydrogen peroxide gel (Total Bleach), Groups 3 and 4 were bleached with Total Bleach modified by the addition of sodium fluoride and calcium chloride, respectively. The microhardness of the enamel surface was assessed using a Vickers microdurometer immediately after the bleaching treatment. The specimens were sectioned in the central portion, polished and evaluated to determine the microhardness of the enamel subsurface to a depth of 125 microm, with an interval of 25 microm between measures. There were significant differences among the groups. In terms of surface microhardness, the bleached group exhibited the lowest means, and the calcium-modified bleached group exhibited the highest means. Regarding subsurface microhardness, there were no significant differences among the groups for the depth and interaction factors. The bleached group exhibited the lowest means, and the calcium-modified bleached group presented the highest means. It was concluded that the bleaching treatment with 35% hydrogen peroxide significantly reduced the surface and subsurface microhardness of the enamel, and the addition of fluoride and calcium in the bleaching agent increased the microhardness means of the bleached enamel.

Identification of Mycobacterium bovis Isolates by a multiplex PCR.

Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates.

Sequencing of hsp65 gene for identification of Mycobacterium species isolated from environmental and clinical sources in Rio de Janeiro, Brazil.

This study evaluated the biodiversity of 28 clinical and 24 environmental Mycobacterium isolates from Rio de Janeiro, Brazil, by using hsp65 sequences, with the aim of contributing to a better understanding of the genetic diversity and usefulness of this marker. An extensive phylogenetic analysis was performed. The nucleotide diversity was similar between clinical (0.06508) and environmental (0.06221) isolates.

Usefulness of Mycobacterium tuberculosis molecular typing in a tuberculosis low-endemic agro-industrial setting of Brazil.

To highlight the transmission and major phylogenetic clades of Mycobacterium tuberculosis, a retrospective study was carried out at two health facilities in a small agro-industrial area in Sao Paulo, Brazil, that has a low tuberculosis incidence rate. IS6110-RFLP and spoligotyping were performed on the isolates, with the former revealing that 31.3% (35/112) of strains were clustered. Epidemiological links were found in 16 of the 35 clustered patients and were associated with transmission among patients living in public housing. Spoligotyping grouped 62.8% of the strains. The T genetic family predominated among the isolates. Of interest is that five strains had a pattern characteristic of African or Asian origin (ST535), and two others were of the rare localized type ST1888 (BRA, VEN). In addition, three new types--1889, 1890, and 1891--were identified. Spoligotyping showed that some ST may be circulating to or from Brazil, and RFLP revealed ongoing transmission in inadequately ventilated public-housing buildings. This may point to a failure in tuberculosis control policy.

An alternative for the preadsorption step in the paratuberculosis serodiagnosis: Mycobacterium fortuitum.

ELISAs for paratuberculosis employ a preadsorption step with Mycobacterium phlei to diminish unspecific reactions As M. fortuitum is one of the most frequent environmental mycobacteria, the purpose of this pilot study was to evaluate its use as an alternative for the preadsorption in ELISAs for paratuberculosis. Results suggest that M. fortuitum can be an alternative instead of or associated to M. phlei with comparable results (κ > 0.8) to conventional ELISAs using M. phlei as a preadsorption antigen.

Clinical evaluation of the microscopic-observation drug-susceptibility assay for detection of tuberculosis.

BACKGROUND: There is an urgent need for low-cost methods for rapid, accurate detection of Mycobacterium tuberculosis in clinical specimens. The microscopic-observation drug-susceptibility (MODS) assay is a relatively low-cost and simple liquid culture method that has been proposed for use in resource-limited environments. METHODS: This prospective study evaluated the performance of the MODS assay for detection of M. tuberculosis in persons undergoing evaluation for pulmonary tuberculosis in Brazil and Honduras. Respiratory specimens were evaluated using smear microscopy, culture on Lowenstein-Jensen medium, and culture using the MODS assay. A subset of specimens was also cultured using the Mycobacterial Growth Indicator Tube (MGIT) 960 automated system (Becton Dickinson). A study subject was considered to have tuberculosis if at least 1 culture on Lowenstein-Jensen medium was positive for M. tuberculosis. FINDINGS: A total of 1639 respiratory specimens obtained from 854 study subjects were analyzed. On a per-subject basis, MODS sensitivity was 97.5% (95% confidence interval [CI], 95.7-98.6), and specificity was 94.4% (95% CI, 93.1-95.2). Median times to detection were 21 days (interquartile range [IQR], 17-25 days) and 7 days (IQR, 5-10) for culture on Lowenstein-Jensen medium and for the MODS assay, respectively (P<.01). For 64 specimens cultured using the MGIT 960 automated system, median time to growth was similar for the MODS assay (7 days; IQR, 7-10 days) and the MGIT 960 automated system (8 days; IQR, 6-11.5 days; P=.16). The percentage of contaminated cultures was lower for the MODS assay than for culture on Lowenstein-Jensen medium (3.8% vs. 5.8%; P<.01). CONCLUSIONS: The MODS assay is a relatively simple test whose good performance characteristics for detection of pulmonary tuberculosis may make it suitable for resource-limited environments.

The level of PPD-specific IFN-gamma-producing CD4+ T cells in the blood predicts the in vivo response to PPD.

There are no reliable means for detecting subclinical mycobacterial infections. The recent sequencing of several mycobacterial genomes has now afforded new opportunities for the development of pathogen-specific diagnostic tests, critical in the context of leprosy and tuberculosis control. In the present study, we applied a multi-parametric flow cytometric analysis that allowed the investigation of T-cell functions in order to define immunological markers that measure previous exposure to mycobacteria. We compared the in vivo response to PPD, the gold standard skin test reagent for measuring previous exposure to Mycobacterium tuberculosis, with in vitro parameters of leukocyte activation in five PPD positive and five PPD negative healthy volunteers. PPD-stimulated peripheral leukocytes expressing CD4, CD69, cutaneous lymphocyte-associated antigen (CLA) and intracellular IFN-gamma were enumerated in whole blood and compared with the size of in vivo PPD-induced induration and IFN-gamma production levels as measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells. The reactivity to the tuberculin skin test (TST) was associated with markedly increased frequencies of PPD-responsive activated (CD69+) and IFN-gamma-producing CD4+T cells. Detection of PPD-specific IFN-gamma producing leukocytes was restricted to CD4+T cells and a subset of these cells was shown to express the skin homing molecule CLA. Multiple linear regression modeling of responses to PPD showed the highest association between skin test indurations and frequencies of PPD-responsive IFN-gamma-producing CD4+CD69+ T cells. Our data show that the in vitro enumeration of antigen-specific IFN-gamma-producing CD4+ T cells can provide an alternative to the in vivo tuberculin test for the detection of latent Mycobacterium tuberculosis infection. Moreover, the measurement of these immunological parameters can be useful for the screening of new specific antigens defined by the genome sequence allowing selection of the best candidates for new diagnostics (including new skin tests), and vaccines for leprosy and tuberculosis.

Diagnosis of paratuberculosis in a dairy herd native to Brazil.

Paratuberculosis (PTB) in Brazil has previously only been reported in imported animals and is officially considered as an exotic disease. A dairy herd, which had no imported animals, presented clinically suspect animals and was investigated for paratuberculosis using faecal culture, histopathology, indirect ELISA and the agar gel immunodiffusion test. Infection with Mycobacterium avium subsp. paratuberculosis (Map) was confirmed by culture of faeces from five cows with clinical symptoms of PTB and in 7/24 randomly selected asymptomatic cows from the same herd. Two cows with clinical symptoms were necropsied and their tissues were positive for Map by culture and histopathology. Twelve asymptomatic, randomly selected cows were positive on ELISA. The results confirmed the presence of PTB in this dairy herd and for the first time demonstrated the disease in a herd of native-bred cattle in Brazil.

Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods.

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR-restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S-23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.

Heterogeneous resistance to vancomycin in Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri clinical strains: characterisation of glycopeptide susceptibility profiles and cell wall thickening.

The population analysis profile (PAP) method as well as analysis of autolytic activity and cellular ultrastructure by transmission electron microscopy (TEM) were used to characterise Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri clinical strains with reduced susceptibility to glycopeptides. All strains showed heterogeneous profiles to vancomycin and teicoplanin by the PAP method. Subpopulations that grew in the presence of high concentrations of each drug were selected from the PAP as derivative strains. Their glycopeptide minimal inhibitory concentrations (MICs) were determined and subsequently all parental and derivative strains were grown in one-half of the MIC of vancomycin or teicoplanin. An increase in cell wall thickness of all derivative strains was seen by TEM, with statistically significant values (P<0.01) compared with their respective parental strains. In general, variable rates of autolysis among the strains were observed. Cell wall thickness is an important factor involved in glycopeptide resistance and, in association with PAP results, confirmed the Brazilian coagulase-negative staphylococci clinical isolates as being heteroresistant to glycopeptides. Detection of these heteroresistant organisms is important in order to achieve more judicious use of vancomycin and teicoplanin in hospitals.

Survey for tuberculosis in an indigenous population of Amazonia: the Suruí of Rondônia, Brazil.

A cross-sectional survey was carried out in 2003 to investigate the epidemiology of tuberculosis (TB) among the Suruí Indians, Brazilian Amazon. A total of 736 subjects (50.7% females) were examined (80% of the total population). TB suspects underwent standardised evaluation for the presence of signs and/or symptoms of active TB, including chest radiography, PPD skin test, sputum microscopy examination for acid-fast bacilli and mycobacterial culture. A Bacillus Calmette-Guerin vaccine scar was detected in 699 individuals (95.0%). Of all the individuals examined, 120 (16.3%) had undergone previous TB treatment (46.7% females). One hundred and nine individuals were assessed as TB suspects (52.3% females). The survey identified six new cases of TB in the Suruí (three men and three women). Five of the six cases came from only two of the ten villages. Mycobacterium tuberculosis colonies grew from 5 (4.6%) samples (only two of which were also smear-positive) and mycobacteria other than M. tuberculosis were isolated from 14 (12.8%) samples. Diagnosis of TB based exclusively on clinical grounds was established only in the case of a 4-year-old girl. Based on this survey, the prevalence of active TB in the sampled group (N=736) was 815.2 per 100000. This study highlights the urgent need to review and strengthen control strategies directed at indigenous peoples in the country, taking into consideration their social, cultural and environmental differences.

A report of mycobacteriosis caused by Mycobacterium marinum in bullfrogs (Rana catesbeiana).

The occurrence of mycobacteriosis caused by Mycobacterium marinum in a commercial breeding farm of bullfrogs (Rana catesbeiana) in Rio de Janeiro, Brazil is described. Ten animals presented skin lesions on the head and extremities. These and 38 other asymptomatic adult animals from various tanks were killed and at necropsy disseminated granulomatous lesions were observed in the 10 clinically affected animals and in 16 (42.1%) of the asymptomatic frogs. Acid-fast bacilli were observed in all smears of the 10 symptomatic frogs and in all but one from the 16 asymptomatic animals with visceral lesions. Ten samples from the 25 positive animals were randomly selected for culture which yielded four isolates of fast-growing (<7 days) mycobacteria. Those purified isolates were characterised by biochemical traditional means as M. marinum. Identification of the strains was confirmed using reverse-phase high-performance liquid chromatography and a polymerase chain reaction (PCR) restriction enzyme analysis assay. It is suggested that M. marinum is an important agent of granulomatous disease in bullfrogs and that infected animals, even when asymptomatic, could act as reservoirs spreading the disease and contaminating other frogs in the farm.

Case report of lymph nodal, hepatic and splenic tuberculosis in an HIV-positive patient.

We describe a case of a male patient, 38 years old, HIV-positive (most recent CD4 count about 259/mm(3)), with abdominal pain, nausea, vomiting, anorexia, weight loss, and vespertine high fever with chills. His hemogram showed normocytic and normochromic anemia, with a high erythrocyte sedimentation rate (ESR) and gross granulations in the neutrophils. Transaminases were normal. Bone marrow biopsy evidenced a chronic disease anemia pattern and a lack of infectious agents. Abdominal ultrasound examination showed a normal-size spleen, which exhibited heterogeneous parenchyma and multiple small hypoechoic images, together with small ascites, peripancreatic and para-aortic lymphadenopathy. These findings were confirmed by abdominal CT. The liver was normal in size, but had a hyperechoic image, which was not visualized on CT. Histopathological analysis of one of the multiple abdominal lymph nodes obtained by laparoscopic biopsy exhibited a chronic granulomatous inflammatory process, with caseous necrosis. Tissue sections were positive for BAAR (acid-alcohol-resistant bacillus), and the cultures were positive for Mycobacterium tuberculosis. Anti-tuberculosis treatment was begun, and the patient evolved with improvement of his general state, fever remission and weight gain. Splenic tuberculosis is a rare disease, occurring predominantly in patients in late stages of AIDS and/or disseminated tuberculosis. It is a difficult diagnosis, since there are no specific findings. Hence, complementary examinations, such as abdominal ultrasound/ CT, or fine needle aspiration, are usually necessary for investigation and differential diagnosis. Often, lesion regression after anti-tuberculosis regimens can be seen, and splenectomy is restricted to complicated or refractory disease.

Multilocus enzyme electrophoresis analysis of rapidly-growing mycobacteria: an alternative tool for identification and typing.

OBJECTIVES: Rapidly growing mycobacteria (RGM) have emerged as important pathogens in clinical settings, associated with esthetic procedures and postsurgical infections, pulmonary infections among cystic fibrosis patients, and other structural pulmonary diseases. Microorganisms belonging to Mycobacterium abscessus-Mycobacterium chelonae and to Mycobacterium fortuitum groups have frequently been associated with outbreaks and various epidemics. In the present study, RGM strains were characterized in order to investigate molecular markers based on proteomic analysis. METHODS: Multilocus enzyme electrophoresis (MLEE) was used for species identification and clonal analysis of RGM recovered from postsurgical wound infections during an epidemic. The study included 30M. abscessus subsp. bolletii clinical isolates, most belonging to the BRA100 clone (epidemic in Rio de Janeiro city), as well as 16 RGM ATCC reference strains. RESULTS: Molecular typing allowed the detection of diversity in the studied population and revealed species-specific isoenzymatic patterns. Additionally, the clonal relationship among M. abscessus subsp. bolletii outbreak isolates, as examined using MLEE, was markedly consistent. CONCLUSIONS: Isoenzymatic characterization was found to be a useful molecular tool to identify RGM species and to determine the relatedness among closely related M. abscessus subsp. bolletii isolates. This may be considered a powerful approach for epidemiological studies on RGM.

Assessment of TerraClass and MapBiomas data on legend and map agreement for the Brazilian Amazon biome

Reliable environmental monitoring and evaluation require high-quality maps of land use and land cover. For the Amazon biome, the TerraClass and MapBiomas projects apply different methodologies to create these maps. We evaluated the agreement between land cover and land use maps generated by TerraClass and MapBiomas (Collections 2 and 3) for the Brazilian Amazon biome, from 2004 to 2014. Specifically, we: (1) described both project legends based on the LCCS (Land Cover Classification System); (2) analyzed the differences between their classes; and (3) compared the mapping differences among the Brazilian states that are totally or partially covered by the Amazon biome. We compared the classifications with a per-pixel approach and performed an evaluation based on agreement matrices. The overall agreement between the projects was 87.4% (TerraClass x MapBiomas 2) and 92.0% (TerraClass x MapBiomas 3). We analyzed methodological differences to explain the disagreements in class identification. We conclude that using these maps together without a properly adapted legend is not recommended for the analysis of land use and land cover change. Depending on the application, one mapping system may be more suitable than the other. (AU)

Liquid vs Solid Culture Medium to Evaluate Proportion and Time to Change in Management of Suspects of Tuberculosis-A Pragmatic Randomized Trial in Secondary and Tertiary Health Care Units in Brazil.

BACKGROUND: The use of liquid medium (MGIT960) for tuberculosis (TB) diagnosis was recommended by WHO in 2007. However, there has been no evaluation of its effectiveness on clinically important outcomes. METHODS AND FINDINGS: A pragmatic trial was carried out in a tertiary hospital and a secondary health care unit in Rio de Janeiro City, Brazil. Participants were 16 years or older, suspected of having TB. They were excluded if only cerebral spinal fluid or blood specimens were available for analysis. MGIT960 technique was compared with the Lowenstein-Jensen (LJ) method for laboratory diagnosis of active TB. Primary outcome was the proportion of patients who had their initial medical management changed within 2 months after randomisation. Secondary outcomes were: mean time for changing the procedure, patient satisfaction with the overall treatment and adverse events. Data were analysed by intention-to-treat. Between April 2008 and September 2011, 693 patients were enrolled (348 to MGIT, 345 to LJ). Smear and culture results were positive for 10% and 15.7% of participants, respectively. Patients in the MGIT arm had their initial medical management changed more frequently than those in the LJ group (10.1% MGIT vs 3.8% LJ, RR 2.67 95% CI 1.44-.96, p = 0.002, NNT 16, 95% CI 10-39). Mean time for changing the initial procedure was greater in LJ group at both sites: 20.0 and 29.6 days in MGIT group and 52.2 and 64.3 in LJ group (MD 33.5, 95% CI 30.6-36.4, p = 0.0001). No other important differences were observed. CONCLUSIONS: This study suggests that opting for the MGIT960 system for TB diagnosis provides a promising case management model for improving the quality of care and control of TB. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN79888843.