Mycobacteria. General culture methodology and safety considerations.
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Animals↗
Biomedical subjects
B W Allen
Publications and source records attributed to B W Allen.
Metronidazole has no antibacterial effect in Cornell model murine tuberculosis.
SETTING: Experiments in vitro on the bactericidal activity of metronidazole and in the Cornell model of murine tuberculosis. OBJECTIVE: To assess the sterilising activity of maximal metronidazole dosage and its activity against bacilli held dormant by immunity in the mouse. DESIGN: In vitro experiments showed that metronidazole was only bactericidal at attainable concentrations (50-100 microg/ml) under anaerobic conditions. In the Cornell model, isoniazid 25 mg/kg and high dosage pyrazinamide 1000 mg/kg was given in the diet with and without 1500 mg/kg metronidazole for the initial 14 weeks of sterilising chemotherapy. In the subsequent sterile state, metronidazole at 0, 100 and 250 mg/kg was given by daily gavage for 6 weeks. Finally, the mice were given 3 weeks of high dosage steroids and their organs were cultured in selective liquid medium. RESULTS: Metronidazole had no activity either in the initial sterilising phase or in the subsequent sterile state. CONCLUSION: The O2 tension in the cellular lesions of murine tuberculosis is unlikely to be sufficiently low to allow metronidazole to act. Its activity should be assessed in caseous lesions.
Animals↗
Enzymatically amplified voltammetric sensor for microliter sample volumes of salicylate.
A new voltammetric sensing strategy for salicylate employing two enzymes and applicable to microliter sample volumes is demonstrated. The method involves the use of the enzyme salicylate hydroxylase to convert salicylate to catechol, which is oxidized at a carbon electrode. The product of this oxidation reaction, o-quinone, is then reduced by a second enzyme, glucose oxidase, to regenerate catechol. Reoxidation of catechol results in a signal that is amplified due to repeated cycling of catechol molecules between the oxidized and reduced states. This chemistry is implemented in two configurations. (i) A paper disk into which both enzymes have been absorbed is mounted on a coplanar three-electrode assembly for aqueous experiments. Determination of salicylate in a nonprescription dermatological product is demonstrated. (ii) A small solution volume confined directly on the coplanar electrodes is used for determination of salicylate in whole blood. The advantages of the use of two enzymes and of monitoring steady-state catalytic currents are discussed.
Electrochemistry↗
Gastro-intestinal involvement in Mycobacterium avium-intracellulare infection of patients with HIV.
In a study of 866 faecal specimens from 437 persons, Mycobacterium avium-intracellulare (MAI) was isolated from 14.8% patients with AIDS and 1.3% patients with symptomatic HIV infection but not from any HIV seronegative or asymptomatic HIV seropositive persons. These data support the hypothesis that the gastro-intestinal tract is the portal of entry for MAI and confirm that MAI infection is a manifestation of late-stage HIV disease. Positive faecal cultures correlated well with disseminated disease. The use of faecal cultures for early diagnosis is therefore recommended.
AIDS-Related Opportunistic Infections↗
Negative sputum smear results in HIV-positive patients with pulmonary tuberculosis in Lusaka, Zambia.
During recruitment to a prospective study of tuberculosis patients in Lusaka, Zambia, 109 had pulmonary disease proven by sputum culture for Mycobacterium tuberculosis, of whom 72 were HIV-1 antibody-positive and 37 were HIV-negative. Among these culture-proven cases, 43% of the HIV-positive patients had a negative sputum smear, compared with 24% of the HIV-negative cases. There was a strong trend towards lower grade or negative sputum smear in the HIV-positive group (P = 0.003). HIV-positive cases also had lower colony counts on culture and colonies took longer to appear. The findings imply that cases of HIV-associated pulmonary tuberculosis may frequently be missed and emphasise the need for new diagnostic methods.
AIDS-Related Opportunistic Infections↗
Application of DNA fingerprinting with IS986 to sequential mycobacterial isolates obtained from pulmonary tuberculosis patients in Hong Kong before, during and after short-course chemotherapy.
A total of 266 Mycobacterium tuberculosis isolates were subjected to DNA RFLP analysis. They were obtained from monthly sputum cultures from patients treated with short-course chemotherapy and then followed up for 2 years. They originated from 42 patients who relapsed after short-course chemotherapy and from a further 42 patients who yielded a single isolated positive culture after chemotherapy. The isolates consisted of one obtained pretreatment and the last obtained during chemotherapy, together with either two isolates cultured at least 2 months apart during relapse or the single post-chemotherapy isolate. They were coded before DNA RFLP analysis and assigned to groups with identical or near identical band patterns on visual inspection. After decoding, it was evident that almost every patient was infected with a strain with a different band pattern (fingerprint). In 100 comparisons of either the pretreatment isolate against the last positive isolate obtained during chemotherapy, or of the first relapse isolate against the second relapse isolate, 15 had been recorded as different; 4 of these were retrospectively found to be due to reading error (error rate 1.5%), leaving 11 (11%) with marked differences. For 5 (12%) of the 42 patients who relapsed, the fingerprint of the relapse isolate was markedly different from that of the pretreatment isolate. In contrast, the isolated positive culture was markedly different from that initially present in 36 (90%) of 40 comparisons. The relative contributions by clinical mixed infection and laboratory cross-contamination to the remaining 10-12% discrepancy rates could not be assessed.(ABSTRACT TRUNCATED AT 250 WORDS)
Antitubercular Agents↗
Quantitative bacillary response to treatment in HIV-associated pulmonary tuberculosis.
A group of 122 patients with culture-proven pulmonary tuberculosis were recruited to examine the concentrations of Mycobacterium tuberculosis in sputum and the relationship to HIV-1 antibody status. They were followed for up to 28 days from the start of antituberculous chemotherapy to assess the early bacillary response to two chemotherapeutic regimens. Of 67 treated with streptomycin, thiacetazone, and isoniazid 17 were HIV positive, and subsequently 55, of whom 20 were HIV positive, were treated with streptomycin, rifampin, isoniazid, and pyrazinamide. The mean initial concentration of M. tuberculosis in the sputum of the HIV-negative patients was significantly higher than in HIV-positive patients (6.95 and 6.34 log colony-forming units respectively; p = 0.019). The HIV-positive patients had less radiologic evidence of disease and significantly fewer zones of lung affected with cavities. The response to treatment was similar, but with HIV-positive patients more likely to become culture negative by 28 days. The differences that exist between HIV-positive and HIV-negative patients are minor, and standard regimens are at least as effective in HIV-positive patients in the first month of treatment.
AIDS-Related Opportunistic Infections↗
Cerebrospinal fluid drug concentrations and the treatment of tuberculous meningitis.
Tuberculous meningitis is a very serious form of tuberculosis. In the absence of randomized controlled trials of alternative treatment regimens, its management depends on employing potent drugs that penetrate well into the cerebrospinal fluid (CSF). The penetration of isoniazid, rifampin, and streptomycin into the CSF of 27 Chinese patients was studied using fluorimetric and microbiologic procedures. Isoniazid rapidly diffused into the CSF, peak concentrations in excess of 3 mg/L, or over 30 times its minimal inhibitory concentration (MIC) against Mycobacterium tuberculosis being attained within 4 hr. In contrast, rifampin and streptomycin penetrated very slowly across the meninges, and CSF levels only slightly in excess of their MICs against M. tuberculosis were achieved. The penetration of the drugs into the CSF correlated poorly with differences in their partitioning between octanol/water and cyclohexane/water but could be predicted using a simple model based on their renal clearance rates and plasma protein binding. It is recommended that patients with tuberculous meningitis should be treated for at least 9 months with a combination of isoniazid, rifampin, and pyrazinamide, which may be supplemented in the first 2 mo with streptomycin.
Administration, Oral↗
The early bactericidal activity of rifabutin measured by sputum viable counts in Hong Kong patients with pulmonary tuberculosis.
Previously untreated patients with smear-positive pulmonary tuberculosis were randomly allocated to treatment with 600, 300, 150 or 75 mg doses of rifabutin (LM427, ansamycin), 600, 300 or 150 mg of rifampicin, 300 mg isoniazid or to no drug daily for 2 days. The fall in viable counts of Mycobacterium tuberculosis in sputum collections during the 2 days, termed the early bactericidal activity (EBA), was estimated from counts of colony-forming units (cfu) on selective 7H-11 agar medium. The EBA for rifabutin ranged from -0.039 (an increase in counts) to 0.049 log10 cfu/ml/day whereas the EBA increased from 0.071 for 150 mg rifampicin to 0.293 log10 cfu/ml/day for 600 mg rifampicin and was 0.43 log10 cfu/ml/day for 300 mg isoniazid. The difference between the EBAs for rifabutin and rifampicin just attained significance (P = 0.05) suggesting that rifabutin was inactive or less active than rifampicin against the extracellular bacilli in pulmonary cavities. Peak plasma concentrations of rifabutin after the initial doses were found to be proportional to dose size and were approximately 7 times lower than those after the same dose size of rifampicin. The lower EBA of rifabutin as compared to rifampicin is probably due to the low plasma concentrations which are not fully compensated for by slightly greater antituberculosis activity of rifabutin in vitro.
Adolescent↗
Counts of viable tubercle bacilli in sputum related to smear and culture gradings.
Pairs of sputum specimens obtained pre-treatment from 166 smear-positive patients with pulmonary tuberculosis were examined by direct smear, culture on Löwenstein-Jensen medium after decontamination by the Petroff method, and by quantitative colony counting on selective 7H11 medium after digestion with dithiothreitol. The selective medium counts ranged from no growth to 8.3 log10 cfu/ml with the largest numbers in the range 3.5-7.0 log10 cfu/ml. Although there was overlap in counts between specimens with negative and positive direct smears, a specimen with a count of 4.0 log10 cfu/ml or more was likely to have a positive smear while a negative smear was likely if the count was lower. This demarcation value should be increased to 4.5 log10 cfu/ml to account for under-estimation in the selective medium counts. The corresponding demarcation estimate for LJ cultures was 80 colonies. In distinguishing between patients in the bacterial contents of their sputum, the selective medium counts were better than the gradings of either LJ cultures or direct smears.
Bacteriological Techniques↗
Comparison of three methods for decontamination of faeces for isolation of Mycobacterium tuberculosis.
Sputum and faeces were obtained from 276 patients on admission to a study of drug resistance in Hong Kong. Acid-fast bacilli were detected microscopically in 103 (37%) sputum specimens and 135 (49%) yielded Mycobacterium tuberculosis on culture. Three methods were used to decontaminate faeces prior to dilution and culture in selective liquid Kirchner medium. A total of 61 faecal specimens were positive for M. tuberculosis on culture and, of these, pretreatment with sodium hydroxide yielded 60 (98%), Portaels modification of Wolinsky and Rynearsons's method 28 (46%) and the combined use of benzalkonium chloride and 1-hexdecylpyridinium chloride yielded 32 (52%). It is recommended that faeces should be treated with sodium hydroxide followed by dilution and culture in selective media, although it may be necessary to formulate new selective media for mycobacterial species other than M. tuberculosis.
Bacteriological Techniques↗
Slide culture sensitivity tests.
A new method for slide culture sensitivity tests of Mycobacterium tuberculosis is described in which smear-positive sputum spread on slides is incubated without prior decontamination in a selective lysed human blood medium. Results are available 7 days after setting up the tests and are particularly useful for guiding the treatment of smear-positive patients with a long history of unsuccessful chemotherapy. Drug concentrations and definitions of resistance are suggested for tests against isoniazid, streptomycin, PAS, rifampicin ethambutol and ethionamide. A good correlation was seen between the results of these tests and those of standard indirect sensitivity tests.
Antitubercular Agents↗
Isolation of Mycobacterium tuberculosis from faeces.
Faeces from patients with Myco, tuberculosis in their sputum was decontaminated using NaOH, acid and an alkaline precipitation method. Treated specimens were diluted, to reduce the number of surviving normal flora, and cultured in liquid selective media. Sub-cultures were made on selective agar and Lowenstein-Jensen medium. Both alkaline methods were superior to acid treatment. Use of all three methods, dilution and culture, yielded growth of Myco, tuberculosis from 90% of patients' faeces, although previous reports have cited only 25-30% of patients excreting bacilli in their faeces.
Bacteriological Techniques↗
Controlled clinical trial of complete open surgical drainage and of prednisolone in treatment of tuberculous pericardial effusion in Transkei.
240 patients with active tuberculous pericardial effusion received a 4-drug daily antituberculosis regimen for 6 months and have been studied for 24 months or longer. Those willing were randomly allocated to open pericardial biopsy and complete drainage of pericardial fluid on admission or percutaneous pericardiocentesis as required. All patients were randomly allocated to prednisolone or matching placebo for the first 11 weeks, on a double-blind basis. Complete open drainage on admission abolished the need for pericardiocentesis (p less than 0.01) but did not influence the need for pericardiectomy for subsequent constriction or the risk of death. Among patients who did not have open drainage on admission, 2 (3%) of 76 given prednisolone compared with 10 (14%) of 74 given placebo died of pericarditis (p less than 0.05), 6 (8%) and 9 (12%) respectively required pericardiectomy, 7 (9%) and 17 (23%) repeat pericardiocentesis (p less than 0.05), and 3 (4%) and 7 (9%) open surgical drainage. By 24 months, apart from the 16 who died from pericarditis, all but 3 patients (2%) had a favourable status.
Adolescent↗
Excretion of viable tubercle bacilli by Blatta orientalis (the oriental cockroach) following ingestion of heat-fixed sputum smears: a laboratory investigation.
Adult Blatta orientalis were allowed to feed on heat-fixed tuberculous sputum smears and the faeces collected for examination by microscopy and culture. Mycobacterium tuberculosis was repeatedly isolated from homogenized faecal pellets using liquid and solid selective culture media. Faeces remained positive both microscopically and on culture even after storing for 8 weeks at room temperature. It is recommended that smears, prepared from clinical material which may contain M. tuberculosis or M. leprae, are stored in a closed container and not left exposed to nocturnal omnivorous insects which frequently infest hospitals and laboratories.
Animals↗
Pathophysiology of pH and Ca2+ in bloodstream and brain.
The highlights of the literature and our work on tetany and hyperventilation are reviewed. Our studies concern the following: (1) the changes of [Ca2+] in circulating plasma caused by respiratory and "metabolic" acidosis and alkalosis; (2) critical plasma [Ca2+] levels associated with signs of tetany and neuromuscular blockade; (3) changes in cerebral [Ca2+]o caused by hypo- and hyper-calcaemia, and the changes in cerebral [Ca2+]o and pHo caused by acute systemic acidosis and alkalosis; and (4) effects of changing [Ca2+]o and pHo levels on synaptic transmission in hippocampal formation. Our main conclusions are (1) changes of plasma [Ca2+] caused by "metabolic" pH changes are greater than those associated with varying CO2 concentration; (2) acute systemic [Ca2+] changes are associated with small cerebral [Ca2+]o changes; (3) the decreases in systemic and cerebral [Ca2+]o caused by hyperventilation are too small to account for the signs and symptoms of hypocapnic tetany; (4) moderate decrease of [Ca2+]o depresses and its increase enhances synaptic transmission in hippocampal formation; and (5) H+ ions in extracellular fluid have a weak depressant effect on neuronal excitability. CO2 is a strong depressant, which is only partly explained by the acidity of its solution. CO2 concentration is a significant factor in controlling cerebral function.
Animals↗
The bioavailability of isoniazid, rifampin, and pyrazinamide in two commercially available combined formulations designed for use in the short-course treatment of tuberculosis.
The bioavailability of isoniazid, rifampin, and pyrazinamide in 2 combined formulations of the 3 drugs (Rifater) for use primarily in the short-course chemotherapy of tuberculosis has been studied in Chinese patients in Singapore and Hong Kong. One formulation, containing 50 mg isoniazid, 120 mg rifampin, and 300 mg pyrazinamide per tablet is suitable for daily use, whereas the other, containing higher proportions of isoniazid and pyrazinamide, is designed for intermittent treatment, each tablet containing 125 mg isoniazid, 100 mg rifampin, and 375 mg pyrazinamide. Appropriate dosages for the Chinese patients, whose average weight was approximately 50 kg, were 5 and 6 tablets, respectively. Plasma concentrations of the 3 drugs after giving such dosages of the 2 combined formulations were compared in 16 patients, 8 in Singapore and 8 in Hong Kong, by means of a crossover study, with the concentrations obtained when identical doses of the 3 drugs were given using standard separate drug formulations. The concomitant urinary excretions of the drugs and their major metabolites were also estimated. Very similar results were obtained whether the drugs were given as the combined preparations or in their standard separate formulations, demonstrating the excellent bioavailability of all 3 drugs in each of the 2 combined formulations.
Acetylation↗
Storage of mycobacteria at -20 degrees C.
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Bacteriological Techniques↗