Potential of Casiopeínas® Copper Complexes and Antituberculosis Drug Combination against Mycobacterium tuberculosis.

New compounds with antituberculosis activity and their combination with classic drugs have been evaluated to determine possible interactions and antagonism. The aim of this study was to evaluate the in vitro activity of Casiopeínas® copper-based compounds (CasIIIia, CasIIIEa, and CasIIgly) alone and combined with isoniazid (INH), rifampicin, or ethambutol (EMB) against resistant and susceptible Mycobacterium tuberculosis. Seventeen clinical M. tuberculosis isolates (5 multi-drug resistant and 2 resistant to INH and/or EMB) were subjected to determination of the minimal inhibitory concentration (MIC) by the resazurin microtiter assay and combination assessment by the resazurin drug combination microtiter assay. The Casiopeínas® alone showed a remarkable effect against resistant isolates with MIC values from 0.78 to 12.50 µg/ml. Furthermore, a synergistic effect mainly with EMB is shown for both resistant and susceptible clinical isolates. Casiopeínas® are promising candidates for future investigation into the development of antituberculosis drugs, being one of the first examples of essential metal-based drugs used in this field.

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Tuberculosis diagnosis after bleach processing for early stage tuberculosis laboratory capacity building.

The diagnosis of tuberculosis is seriously hampered in the absence of standard biosafety laboratory facilities for specimen concentration and Mycobacterium tuberculosis culture. Within a laboratory twinning arrangement, heat-fixed direct smear and sediment from 74 bleach-processed and 20 non-processed specimens from Cumura Hospital, Guinea-Bissau, were sent to Lisbon for molecular evaluation of rifampicin resistance. Sequence analysis of a 369 base-pair rpoB locus detected 3.2% (3/94) resistant specimens. To our knowledge, this represents the first report on the molecular analysis of M. tuberculosis from bleach-processed sputum, an alternative to current diagnostic practice in low-resource settings.

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide.

The microscopic observation drug susceptibility assay (MODS) was evaluated to determine susceptibility to pyrazinamide in Mycobacterium tuberculosis, and compared with the broth microdilution method (BMM), absolute concentration method (ACM), and pyrazinamidase (PZase) determination. We tested 34 M. tuberculosis clinical isolates (24 sensitive and eight resistant to pyrazinamide) and the control strains M. tuberculosis H37Rv (ATCC 27294) and Mycobacterium bovis AN5. The MODS, BMM, ACM and PZase determination provided results in average times of 6, 18, 28 and 7 days, respectively. All methods showed excellent sensitivity and specificity (p <0.05). Of the methods studied, the MODS proved to be faster, efficient, inexpensive, and easy to perform. However, additional studies evaluating the MODS in differentiating pyrazinamide-resistant and pyrazinamide-susceptible M. tuberculosis must be conducted with a larger number of clinical isolates.

Byrsonima fagifolia Niedenzu Apolar Compounds with Antitubercular Activity.

Bioassay-guided fractionation of the chloroform extract of Byrsonima fagifolia leaves led to the isolation of active antitubercular compounds alkane dotriacontane (Minimal Inhibitory Concentration-MIC, 62.5 µg mL(-1)), triterpenoids as bassic acid (MIC = 2.5 µg mL(-1)), α-amyrin acetate (MIC = 62.5 µg mL(-1)), a mixture of lupeol, α- and ß-amyrin (MIC = 31.5 µg mL(-1)) and a mixture of lupeol, and acetates of α- and ß-amyrin (MIC = 31.5 µg mL(-1)). The antimycobacterial activity was determined by the Microplate Alamar Blue Assay (MABA) and the structures of promising compounds were determined by spectroscopic analysis. This investigation constitutes the first report of a chemical and antitubercular study of apolar compounds from B. fagifolia Niedenzu (IK).

Antimycobacterial activity of lichen substances.

We describe here the extraction and identification of several classes of phenolic compounds from the lichens Parmotrema dilatatum (Vain.) Hale, Parmotrema tinctorum (Nyl.) Hale, Pseudoparmelia sphaerospora (Nyl.) Hale and Usnea subcavata (Motyka) and determined their anti-tubercular activity. The depsides (atranorin, diffractaic and lecanoric acids), depsidones (protocetraric, salazinic, hypostictic and norstictic acids), xanthones (lichexanthone and secalonic acid), and usnic acid, as well seven orsellinic acid esters, five salazinic acid 8',9'-O-alkyl derivatives and four lichexanthone derivatives, were evaluated for their activity against Mycobacterium tuberculosis. Diffractaic acid was the most active compound (MIC value 15.6mug/ml, 41.6 microM), followed by norstictic acid (MIC value 62.5 microg/ml, 168 microM) and usnic acid (MIC value 62.5 microg/ml, 182 microM). Hypostictic acid (MIC value 94.0 microg/ml, 251 microM) and protocetraric acid (MIC value 125 microg/ml, 334 microM) showed moderate inhibitory activity. The other compounds showed lower inhibitory activity on the growth of M. tuberculosis, varying from MIC values of 250 to 1370 microM.

Detection of nontuberculous mycobacteria from water buffalo raw milk in Brazil.

Milk is an important nutritional source to man and water buffalo raw milk is used to produce mozzarella cheese. Products from unpasteurized milk have been associated with certain infectious diseases and can carry pathogenic mycobacteria. Nontuberculous mycobacteria (NTM) are emerging pathogens causing opportunistic infections in humans and animals. The objectives of this study were to demonstrate the presence of mycobacteria in water buffaloes' milk and to determine their role as possible sources of NTM infections. In this study, raw milk samples from dairy water buffaloes (Bubalus bubalis) (N = 23) were decontaminated by Petroff method and inoculated on to Löwenstein-Jensen and Stonebrink medium. After confirming positive colonies for acid fast bacilli (AFB) by Ziehl-Neelsen technique, the isolated mycobacteria were identified by PCR-Restriction Enzyme Analysis (PRA) and mycolic acids analysis by thin-layer chromatography (TLC). Mycobacterium simiae (2 isolates), Mycobacterium kansasii (2 isolates), Mycobacterium flavescens (2 isolates), Mycobacterium gordonae (3 isolates) and Mycobacterium lentiflavum (1 isolate) were identified by these techniques. The isolation of opportunistic pathogens such as M. kansasii, M. simiae and M. lentiflavum from raw milk represent a risk for the consumers of mozzarella cheese made by this milk.

Direct detection of Mycobacterium bovis in bovine lymph nodes by PCR.

Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff's method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 microl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius.

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.

Antimycobacterial activity of some Brazilian indigenous medicinal drinks

Tuberculosis (TB) is a very serious problem worldwide and the increasing number of multiple drugs resistant TB cases makes the search for new anti-TB drugs an urgent need. Indigenous knowledge about the use of native plants to treat illnesses has contributed to the discovery of new medicines. In this study, the antimycobacterial activity ofseven medicinal drinks was assessed: Ananas sativus (hydroalcoholic fruit extract), Aristolochia triangularis(aqueous and hydroalcoholic leaf, root and stem extracts), Bromelia antiacantha (hydroalcoholic fruit extract), Stryphnodendron adstringens (hydroalcoholic bark extract), Tabebuia ovellanedae (hydroalcoholic bark extract), Vernonia polyanthes (hydroalcoholic root extract), all used by the Vanuíre indigenous community in the treatment of respiratory diseases. The activity was evaluated by using a time-to-kill assay, in which Mycobacterium tuberculosis H37Rv was cultured on Lowenstein-Jensen medium, after thirty minutes, one, three, six, twelve and twenty-four hours contact of the bacteria with each drink. Within half to one hour contact, the hydroalcoholic drinks of A. triangularis, S. adstringens, T. ovellanedae and V. polyanthes reduced the bacterial growth by 2 orders of magnitude in CFU/mL, and all bacterial growth was absent after three hours contact. In contrast, no mycobactericidal effect was detected in the aqueous extract of A. triangularis or in the hydroalcoholic beverages of A. sativus and B. antiacantha, even aftertwenty-four hours contact.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil.

OBJECTIVES: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined. METHODS: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products. RESULTS: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity. CONCLUSIONS: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

A duplicated nitrotienyl derivative with antimycobacterial activity: synthesis, X-ray crystallography, biological and mutagenic activity tests.

A duplicated nitrotienyl derivative was obtained as a by-product from the synthesis of a proposed molecular hybrid of a nitrotienyl derivative and isoniazid with an expected dual antimycobacteria mechanism. The structure was shown to be the 5,5'-dinitro-2-(2,3-diaza-4-(2'-tienyl)buta-1,3-dienyl)tiophene by X-ray crystallography. The minimal inhibitory concentration (MIC) determination of this compound proved to be promising against Mycobacterium pathogenic strains such as M. avium and M. kansasii, although it had a high level of mutagenicity, as observed in mutagenic activity tests.

Rapid molecular diagnosis of tuberculosis and other mycobacteriosis in smear-negative clinical specimens

Delay in diagnosis of pulmonary and other forms of tuberculosis (TB) can be fatal, particularly in HIV-infected patients. Hence, techniques based on nucleic acid amplification, which are both rapid and of high specificity and sensitivity, are now widely used and recommended for laboratories that diagnose TB. In the present study, diagnostic methods based on mycobacterial DNA amplification were evaluated in comparative trials alongside tradicional bacterial methods, using negative smear samples from patients with clinically-suspected TB (sputum samples from 25 patients with suspected pulmonary TB, urine samples from two patients with suspected renal TB and cerebrospinal fluid samples from one patient with suspected meningeal TB). A specificity of 100% was achieved with DNA amplification methods and tradicional culture/identification methods, in relation to clinical findings and treatment results. For the smear-negative sputa, conventional PCR for M.tuberculosis was positive in 62% of suspected lung TB case, showing the same sensitivity as bacterial identification. Both techniques failed in the detection of extra-pulmonary samples. Nested PCR showed, after species-specific amplification, a sensitivity of 100% for M. avium and 85% for M. tuberculosis. For extra-pulmonary smear-negative samples, only Nested PCR detected M. tuberculosis and all cases were confirmed clinically. Nested PCR, in which two-step amplification reactions are performed, can identify the two most important mycobacteria in human pathology quickly and directly from clinical spicimens

Isolation of Mycobacterium gordonae from poultry slaughterhouse water in São Paulo State, Brazil.

Water samples (24 untreated water, 12 treated water and 24 served water) used in different stages of the slaughter process were examined to identify a possible source of pathogenic mycobacteria. The isolates were identified based on microscopy, morphological and biochemical features, mycolic acid analysis and molecular method--PCR-restriction-enzyme analysis. Eighteen mycobacterial strains were isolated from 60 water samples: 11 from untreated water, 5 from treated water and 2 from served water. All mycobacteria isolated were identified as Mycobacterium gordonae and showed the following PRA genotypes: III (27.8%), IV (38.9%) and V (33.3%).

Isolation of Mycobacterium gordonae from poultry slaughterhouse water in SÒo Paulo State, Brazil

Water samples (24 untreated water, 12 treated water and 24 served water) used in different stages of the slaughter process were examined to identify a possible source of pathogenic mycobacteria. The isolates were identified based on microscopy, morphological and biochemical features, mycolic acid analysis and molecular method--PCR-restriction-enzyme analysis. Eighteen mycobacterial strains were isolated from 60 water samples: 11 from untreated water, 5 from treated water and 2 from served water. All mycobacteria isolated were identified as Mycobacterium gordonae and showed the following PRA genotypes: III (27.8

) and V (33.3

Caracterização da população portadora de tuberculose do município de Américo Brasiliense/SP, no período de 1992 a 2002 / Caracterization of tuberculosis patients of Americo Brasilienses's city on 1992 to 2002

A tuberculose é um grave problema de Saúde Pública em todo o mundo, sendo desde 1993 considerada pela OMS uma emergência global. O Brasil ocupa a 13ª posição no ranking mundial em incidência de tuberculose. Ocorrências de tuberculose nas grandes cidades brasileiras são bem conhecidas e estudadas. Entretanto, pouco se sabe sobre a problemática da tuberculose nas pequenas cidades. Neste sentido, este trabalho visou obter as características epidemiológicas da população portadora de tuberculose do município de Américo Brasiliense, SP, no período de 1992 a 2002. Os resultados mostraram que a incidência de tuberculose nesta cidade teve picos coincidentes com os anos de intensa migração de mão de obra, sobretudo para o corte da cana de açúcar e que a tuberculose acomete principalmente os trabalhadores rurais do sexo masculino, na idade produtiva dos 20 aos 40 anos. Foi verificado também que a tuberculose pulmonar é a principal forma clínica e que os índices de abandono de tratamento e de cura, foram respectivamente de 1,8% e aproximadamente, de 90%. O índice de detecção da doença pela baciloscopia foi de cerca de 60%

Plantas do cerrado brasileiro com atividade contra Mycobacterium fortuitum / Brazilian cerrado plants active against Mycobacterium fortuitum

Mycobacterium fortuitum é uma micobactéria de crescimento rápido, ubíquo na natureza e relacionada a micobacteriose de importância médica.Ela tem sido isolada de bacteremias, abscessos, endocardites, feridas cirúrgicas e traumáticas.De difícil tratamento, o bacilo é reconhecido na literatura como resistente inclusive aos medicamentos utilizados na terapêutica da tuberculose.O objetivo deste trabalho foi pesquisar extratos vegetais do Cerrado brasileiro com atividade contra M. fortuitum, empregando a técnica do Microplate Alamar Blue Assay (MABA) como método analítico.Dos 26 extratos testados frente ao M.fortuitum, o extrato apolar de Quassia amara (extrato diclorometanico) foi o que apresentou melhor resultado com valor de CIM de 62,5mg/mL seguidos pelos extratos apolares de Syngonanthus macrolepsis, Davilla elliptica, Turnera ulmifolia com CIM de 125g/mL.Para as mesmas plantas analisadas, utilizando-se agentes extratores polares (etanol e metanol), foram verificados CIM superiores a 500g/mL.Os valores foram semelhantes aos de extratos de outras plantas analisadas sendo considerados não promissores.

Isolation of Mycobacterium gordonae from poultry slaughterhouse water in SÒo Paulo State, Brazil.

Water samples (24 untreated water, 12 treated water and 24 served water) used in different stages of the slaughter process were examined to identify a possible source of pathogenic mycobacteria. The isolates were identified based on microscopy, morphological and biochemical features, mycolic acid analysis and molecular method--PCR-restriction-enzyme analysis. Eighteen mycobacterial strains were isolated from 60 water samples: 11 from untreated water, 5 from treated water and 2 from served water. All mycobacteria isolated were identified as Mycobacterium gordonae and showed the following PRA genotypes: III (27.8

), IV (38.9

) and V (33.3

).