Comparison of MGIT 960 & pyrazinamidase activity assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis.

Background & objectives: Pyrazinamide is an important front line antimycobacterial drug, which is also being used in the treatment of multi drug resistant tuberculosis along with second line drugs in DOTS plus programme. Conventional testing of pyrazinamide on solid medium is difficult as it is active at acidic pH. Therefore, there is a need for a rapid and simple method for susceptibility testing of pyrazinamide. This study was carried out to compare pyrazinamide susceptibility testing by MGIT 960 and two rapid pyrazinamidase activity tests. Methods: Pyrazinamide susceptibility was tested in 136 clinical isolates of Mycobacterium tuberculosis by MGIT 960 and pyrazinamidase activity was tested by classical Wayne’s method and modified PZase agar method. Results: There was 88.9 per cent concordance between MGIT 960 and classical Wayne’s method and 93.38 per cent with modified method for pyrazinamidase activity. Using MGIT 960 results as gold standard the sensitivity and specificity of Wayne’s method was 88.15 and 90 per cent respectively and that of modified method was 89.4 and 98.3 per cent. Interpretation & conclusions: Our study demonstrates that the modified pyrazinamidase activity test can be used as a screening test to detect resistance to pyrazinamide specially in resource limited settings but confirmation of susceptibility should be done by standard methods like MGIT 960.

The paradox of pyrazinamide: an update on the molecular mechanisms of pyrazinamide resistance in Mycobacteria.

Pyrazinamide (PZA) is an important front line anti-tuberculosis drug because of its sterilizing activity against semi-dormant tubercle bacilli. In spite of its remarkable role in shortening the treatment duration from 9 months to 6 months when used in combination with Rifampicin and Isoniazid, PZA remains a difficult paradox because of its incompletely understood mode of action and mechanism of resistance. PZA is a nicotinamide analog prodrug which is converted into the active bactericidal form pyrazinoic acid by the bacterial enzyme pyrazinamidase (PZase). PZA does not appear to have a specific cellular target and instead, exerts its bactericidal effect by disrupting the membrane energetics and acidification of cytoplasm. Majority (72-97%) of PZA-resistant isolates of M. tuberculosis exhibit mutations in their pncA gene or upstream area leading to loss of PZase activity. A wide diversity of pncA mutations scattered along the entire length of pncA gene is unique to PZA resistance. However, PZA resistant isolates with normal PZase activity and wild type pncA sequences have also been reported in several studies which indicate that alternate mechanisms of PZA resistance exist. Investigations into these mechanisms would be useful in developing alternative diagnostic/therapeutic measures. This review presents the update of various mechanisms of PZA resistance in different mycobacteria with special emphasis on mode of action of PZA and mechanisms of resistance in Mycobacterium tuberculosis.

Bioavailability of rifampicin following concomitant administration of ethambutol or isoniazid or pyrazinamide or a combination of the three drugs.

BACKGROUND & OBJECTIVES: Poor bioavailability of rifampicin (R) in combination with other anti-tuberculosis drugs such as isoniazid (H), pyrazinamide (Z), and ethambutol (E) is a subject of much concern for the last few decades. This could be due to an interaction between R and other drugs. An investigation was therefore undertaken to examine the bioavailability of R in the presence of H, Z and E or a combination of the three drugs. METHODS: The study included eight healthy volunteers, each being investigated on four occasions at weekly intervals once with R alone and with three of the four combinations on the three remaining occasions. A partially balanced incomplete block design was employed and the allocation of R or the drug combinations was random. Plasma concentrations of R at intervals up to 12 h were determined by microbiological assay using Staphylococcus aureus as the test organism. The proportion (%) dose of R as R plus desacetyl R (DR) in urine excreted over the periods 0-8 and 8-12 h was also determined. Bioavailability was expressed as an index (BI) of area under time concentration curve (AUC) calculated from the plasma concentrations or proportion of dose of R excreted as R plus DR in urine with the combinations to that with R alone. RESULTS: The bioavailability indices based on AUC were 0.96 with RE, 0.76 with RH, 1.08 with RZ and 0.65 with REHZ. The indices based on urine estimations (0-8 h) were similar, the values being 0.94, 0.84, 0.94 and 0.75, respectively. A second investigation revealed that the decrease of bioavailability of R with H was not due to the excipients present in H tablets. INTERPRETATION & CONCLUSION: Isoniazid alone or in combination with E and Z reduces the bioavailability of R. Urinary excretion data offer a simple and non invasive method for the assessment of bioavailability of R.

Effects of pyrazinamide on pregnant albino rats

Although being used for years in the treatment of tuberculosis, no data are available in the literature on the safety of pyrazinamide during pregnancy. Accordingly, we aimed to make a first approach to this problem by evaluating the effects of this drug administered during the entire pregnancy of albino rats. Fourty female, EPM-1 Wistar albino rats of about 250 g b.w. were used. Upon conception (day zero of pregnancy) the animals were randomly divided in 4 groups of 10 rats each and labeled as follows. Controls (C), animals treated with the drug vehicle (destilled water); experimental groups (E1, E2 and E3), animals treated with 35, 105 or 315 mg/kg b.w. pyrazinamide by gavage (oral route) once daily up to the term (20th day of pregnancy). Drug or vehicle volume was always 0.5 ml. Body weight gain was followed up every week. At term, upon sacrifice (in excess of anesthesia) and histerectomy, the following parameters have been studied: number of implantations and reabsorptions; intrauterine deaths; number of living foetuses and of placentae; weights of concepts and of placentae; major foetal malformations; maternal mortality index. No significant effects of pyrazinamide on rat pregnancy have been observed regarding the maternal body weight gain, the weights of concepts, the number of implantations and reabsorptions and the weights of placentae and foetuses. Also, no deleterious effects have been noticed regarding major foetal malformations, intrauterine deaths or maternal mortality. With the highest pyrazinamide dosis employed (315 mg/kg b.w.), however, a significant lowered uterine weight was recorded. Although otherwise safe, a high-dose regimen of pyrazinamide during rat pregnancy can induce a slight yet significant reduction of uterine weight.

Characterization of pncA Mutations of Pyrazinamide-Resistant Mycobacterium tuberculosis in Korea

Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.

Standartization of broth microdilution method for Mycobacterium tuberculosis

Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100 per cent for all drugs and specifities of 91, 100, 96, 98 and 85 per cent were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days) than the proportional method (20-28 days). The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.

Uveíte tuberculosa: realto de casos atípicos / Tuberculous uveitis: repot of atypcal cases

A tuberculose é a doença epidêmica mais encontrada do Brasil, causada pelo bacilo de Koch, com características granulomatosa e crônica. O diagnóstico ocular é geralmente difícil, especialmente quando os pacientes apresentam exames laboratoriais tais como raios X do tóraz e baciloscopia do escarro normais, restando apenas o teste de Montoux para nos basearmos. Os autores apresentam três casos de uveíte tuberculosa näo relacionada a AIDS, com baixa acuidade visual secundária, sendo por inoculaçäo acidental na pele e outros dois por provável inalaçäo do bacilo. Os pacientes após o diagnóstico receberam tratamento com pirazinamida 2 g (2 meses) + Rifampicina 600 mg (6 meses) + Isoniazida (6 meses) com sensivel melhora do quadro ocular (diminuiçäo da inflamaçäo, melhora da dor e da acuidade visual), caracterizando a etiologia

Lack of effect of cimetidine and ranitidine on pyrazinamide kinetics in rats

Pyrazinamide [25 mgkg-1] kinetics was determined in cimetidine [40 mgkg-1] or ranitidine [50 mgkg-1] pretreated rats for seven days. animals received either 0.9% NaCl solution [control] or cimetidine or raintidine orally [5 mlkg-1]. Disposition of pyrazinamide was studied 24 h after the last does of pretreatment by injecting pyrazinamide [1 mlkg-1] into the tail vein. Blood samples [0.25ml] were drawn at 15.35.45 min. and 1.0, 1.5, 2.0, 3, 4, 5, 6 and 8h respectively after iv administration. Pyrazinamide plasma concentrations were analyzed using HPLC method. Cimetidine and ranitidine had no significant effect on volume of distribution [Vd], half life [t0.5], clearance [CL], mean residence times [MRT] and area under the curves [AUC] of pyrazinamide. These results suggest that cimetidine and ranitidine pretreatment does not affect significantly pyrazinamide kinetics in rats

Efeito da pirazinamida sobre a resposta imune celular humana / Effect of pyrazinamide on human cellular immune response

A incorporaçäo da pirazinamida (PZA) ao esquema quimioterápico da tuberculose permitiu que o seu tratamento fosse encurtado para seis meses. A despeito da eficácia da PZA in vivo, experências in vitro com esta droga näo demonstram atividade bactericida relevante. Com a finalidade de avaliar o efeito da PZA sobre a resposta imune, foram realizados testes cutâneos de hipersensibilidade tardia com PPD e antígeno de Candida albicans ou testes de transformaçäo lifoblástica, em 33 pacientes com tuberculose pulmonar antes e durante o tratamento: 21 em uso de PZA e 12 controles em quimioterapia sem este fármaco. Encontramos transformaçäo linfoblástica basal de 905 ñ 302 cpm nos doentes que usavam PZA, em contraste com 258 ñ 147 cpm anteriores à quimioterapia, e 323 ñ 90 cpm no grupo tratado sem PZA. Estas diferenças säo estatisticamente significantes. Ocorreu também uma tendência de maior reatividade cutânea em pacientes submetidos à PZA, mas com diferenças näo significantes. Nossos resultados podem explicar, em parte, como esta droga age contra o M. tuberculosis e indicam que investigaçäo complementar apropriada deve ser feita para avaliar a possibilidade de emprego da PZA em outras doenças, em que a resposta imunecelular tenha papel importante nos mecanismo de defesa do hospedeiro

Acción de la pirazinamida sobre el hígado de los enfermos tuberculosos: estudio ultraestructural / Effect of pyrazinamide on the liver of tuberculous patients: ultrastructural study

Un grupo de pacientes tuberculosos, varones, adultos, sin enfermedades asociadas, alcoholikas y no alcoholistas, recibieron un esquema terapéutico con isoniacida, rifampicina, estreptomicina y pirazinamida, a las dosis corrientes. Se les efectuó un estudio funcional e histológico del hígado antes del tratamiento y se lo repitió a los 2 meses de recibir las drogas. Un grupo control, estudiado de la misma manera, recibió un esquema terapéutico con isoniacida, rifampicina, estreptomicina y etambutal. Otro grupo de pacientes tuberculosos recibió pirazinamida sola durante 2 semanas y el hígado también fue estudiado antes y después de la quimioterapia. En todos los casos, las muestras hepáticas obtenidas a los 2 meses de recibir drogas asociadas y a las 2 semanas de recibir pirazinamida sola fueron observadas con microscopía electrónica. En los pacientes que recibieron pirazinamida sola el estudio ultraestructural del hígado mostró 3 casos normales y megamitocondrias y/o hiperplasia del retículo endoplásmico liso en los 4 casos restantes. Los pacientes que recibieron un esquema asociado con pirazinamida presentaban a los 2 meses analogas alteraciones hepáticas: megamitocondrias, hiperplasia del retículo endoplásmico liso, solas o asociadas. El grupo control, tratado con un esquema sin pirazinamida, mostró modificaciones ultraestructurales análogas a las del grupo anterior, salvo 1 caso con dilatación del retículo endoplásmico rugoso. En ninguno de los 3 grupos hubo diferencias entre los pacientes alcoholistas y no alcoholistas, y las alteraciones ultraestructurales halladas no tuvieron repercusión clínica ni funcional. Teniendo en cuenta que las alteraciones ultraestructurales del hepatocito observadas en estos pacientes son frecuentes en varias enfermedades hepáticas y que las pueden producir varias drogas incluyendo las drogas antituberculosas, no se las puede considerar privativas de la pirazinamida. Podemos concluir que el empleo de la pirazinamida en el tratamiento de la tuberculosis en pacientes alcoholistas y no alcoholistas, sin enfermedad hepática asociada, y con las característica terapêuticas con que se la maneja actualmente, no aumenta el riesgo hepático que implica el uso de las otras drogas