Biomedical subjects

L Heifets

Publications and source records attributed to L Heifets.

At least 37 records · Page 2Linked to original sources

PubMed1997

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

Amidohydrolases↗

PubMed1996

A randomized trial of clarithromycin as prophylaxis against disseminated Mycobacterium avium complex infection in patients with advanced acquired immunodeficiency syndrome.

BACKGROUND: Disseminated infection with Mycobacterium avium complex is the most common opportunistic infection in patients with advanced stages of the acquired immunodeficiency syndrome (AIDS). We studied the efficacy and safety of prophylactic treatment with clarithromycin, a macrolide antibiotic. METHODS: We conducted a randomized, placebo-controlled, double-blind study of clarithromycin in patients with AIDS in the United States and Europe. Entry criteria included blood cultures that were negative for M. avium complex, a Karnofsky performance score of 50 or higher, a CD4 cell count of 100 or less per cubic millimeter, and a life expectancy of at least six months. RESULTS: After the first interim analysis, the study was stopped. M. avium complex infection developed in 19 of the 333 patients (6 percent) assigned to clarithromycin and in 53 of the 334 (16 percent) assigned to placebo (adjusted hazard ratio, 0.31; 95 percent confidence interval, 0.18 to 0.53; P<0.001). During the follow-up period of about 10 months, 32 percent of the patients in the clarithromycin group died and 41 percent of those in the placebo group died (hazard ratio, 0.75; P=0.026). In the clarithromycin group, isolates from 11 of the 19 patients with M. avium complex infection were resistant to clarithromycin. Prophylaxis with clarithromycin was associated with an increased incidence of taste perversion (11 percent in the clarithromycin group vs. 2 percent in the placebo group, P<0.001) and rectal disorders (8 percent vs. 3 percent, P = 0.007); however, the frequency of more severe adverse events was similar in the two groups (7 percent and 6 percent, respectively). CONCLUSIONS: In patients with advanced AIDS, the prophylactic administration of clarithromycin is well tolerated, prevents M. avium complex infection, and reduces mortality.

AIDS-Related Opportunistic Infections↗

PubMed1996

Molecular mechanisms of clarithromycin resistance in Mycobacterium avium: observation of multiple 23S rDNA mutations in a clonal population.

The peptidyltransferase region of the 23S rRNA gene (the probable target site for the macrolides) was investigated in blood isolates of Mycobacterium avium recovered from 38 patients before and after the development of clarithromycin resistance. Point mutations were identified in 100% of the 74 resistant relapse blood isolates but in none of 69 susceptible pretreatment isolates. Multiple mutations were identified in isolates from 23 (61%) of 38 patients. Of the 63 identified mutations, 95% involved adenine at bp 2058. Single-colony clones from cultures that were mixtures of more than one mutation revealed a single mutation within each clone. Pulsed field gel electrophoresis of genomic DNA restriction fragments revealed that 13 (81%) of 16 multiple mutations identified in the same patient were derived from a single infecting strain. In vitro investigation revealed the same point mutations observed in vivo. This study defines the probable mechanism of clarithromycin resistance in M. avium and provides in vivo evidence that mutational resistance is random and selection-directed.

Base Sequence↗

PubMed1996

Bacteriostatic and bactericidal activities of benzoxazinorifamycin KRM-1648 against Mycobacterium tuberculosis and Mycobacterium avium in human macrophages.

Inhibitory and bactericidal activities of KRM-1648 were determined against Mycobacterium tuberculosis and M. avium residing in human monocyte-derived macrophages and extracellular M. tuberculosis and M. avium. MICs and MBCs of KRM-1648 against intracellular and extracellular bacteria were substantially lower than those of rifampin. The MICs and MBCs of either drug against the intracellular bacteria were only twofold lower than or equal to the values found for extracellular bacteria. The prolonged effect of KRM-1648 found in this study is probably associated with high ratios of intracellular accumulation, which were 50- to 100-fold higher than that found for rifampin. Further studies on intracellular distribution of KRM-1648 and on the sites of actual interaction between the drug and bacteria residing in macrophages are necessary, as well as evaluation of combined effects of KRM-1648 with other drugs in long-term macrophage culture experiments.

Antibiotics, Antitubercular↗

PubMed1996

Susceptibility testing of Mycobacterium avium complex isolates.

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Anti-Bacterial Agents↗

PubMed1995

Comparison of activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human macrophages.

The activities of rifapentine and rifampin against Mycobacterium tuberculosis residing in human monocyte-derived macrophages were determined. The MICs and MBCs of rifapentine for intracellular bacteria were two- to fourfold lower than those of rifampin. For extracellular bacteria, this difference was less noticeable. Nevertheless, the more favorable pharmacokinetics of rifapentine over rifampin was addressed in other experimental models. These models showed substantial differences after short pulsed exposures of the infected macrophages to the drugs and when the infected macrophages were exposed to changing drug concentrations that imitated the pharmacokinetic curves observed in blood. Once-a-week exposures to rifapentine concentrations equivalent to those attained in blood after one 600-mg dose resulted during the first week in a dramatic decline in the number of bacteria, and this decline was maintained at a minimal level for a period of four weeks. The results of this study have shown the suitability of rifapentine for intermittent-treatment regimens. The prolonged effect of rifapentine found in this study may be associated with high ratios of intracellular accumulation, which were four- to fivefold higher than those found for rifampin. Further studies on the intracellular distribution of rifamycins and on the sites of actual interaction between the drugs and bacteria residing in macrophages are necessary.

Antitubercular Agents↗

PubMed1995

Methods for determining concentrations of antimicrobial agents in human monocytes.

Human monocytes can be derived from the leukocyte-rich by-product of donors' blood available after platelet separation. Large volumes of the monocyte samples obtained from this product provided an opportunity to conduct experiments with relatively high concentrations of the antimicrobial agents sufficient for their detection in bioassays, thus avoiding the necessity of working with the radiolabelled drugs. Washing of the cells after their exposure to the drug may lead to an extraction of the tested agent from the cell, especially if it is a substance of low molecular weight. In our experiments we excluded the washing step, and separated the monocytes from the extracellular medium by velocity gradient centrifugation. In experiments with two rifamycins, the cell pellet as well as the extracellular fluid were subjected to a bioassay using Micrococcus luteus as a target organism. The method showed good reproducibility and consistency in results obtained.

Anti-Bacterial Agents↗

PubMed1994

Quantitation of mycobacteria in blood specimens from patients with AIDS.

OBJECTIVE: To determine the effect of a delay in processing blood specimens, that may occur by mailing them to a specialized laboratory, on the quantitation of the number of mycobacteria. DESIGN: Simulated blood specimens in sodium polyanetholium sulfonate (SPS)-containing vacutainers were spiked with Mycobacterium avium, and stored for 4 days after addition of clarithromycin or rifabutin, or remained drug-free. Daily samples were lysed with sodium desoxycholate, and inoculated onto 7H11 agar plates for the subsequent colony-forming units (CFU)/ml count. The isolation rates on two media, 7H11 agar and BACTEC 12B broth, were determined with 1212 specimens from AIDS patients. RESULTS: The number of bacteria in blood specimens remained without substantial change during the period of storage, even in the presence of clarithromycin or rifabutin, especially after refrigeration for 2 or 24 h before storage. 7H11 agar and 12B broth were equally sensitive for isolation of M. avium. CONCLUSIONS: A 4-day delay in processing blood specimens in SPS vacutainers does not affect the results of quantitation of the number of mycobacteria. Combined use of 12B broth and 7H11 agar increased the overall recovery along with an opportunity to quantitate the number of CFU/ml.

AIDS-Related Opportunistic Infections↗

PubMed1994

Effects of clarithromycin and rifabutin alone and in combination on intracellular and extracellular replication of Mycobacterium avium.

The combined effect of clarithromycin and rifabutin against Mycobacterium avium multiplying either within human monocyte-derived macrophages or extracellularly in a liquid medium was additive: both MICs and MBCs were twofold lower for the combination than they were for each drug alone. Prolonged exposure for 4 weeks of M. avium-infected macrophages to combined concentrations that were only twofold greater than the MICs resulted in a 100-fold decrease in the number of viable bacteria, while in the drug-free controls a 100-fold or greater increase in comparison with the initial viable counts took place. Comparison of this effect with the results of the prolonged exposure to each drug alone suggested that under these experimental conditions rifabutin enhanced the antimicrobial activity of clarithromycin against intracellular bacteria. At the same time, inhibition of intracellular growth by a 2-h pulsed exposure of the infected macrophages to the combination of the two drugs was not different from the effect induced by clarithromycin alone. In conclusion, clarithromycin played the major role in the antimicrobial activity of the tested combination, while rifabutin may have enhanced this effect during a prolonged exposure of the intracellular bacteria to these two agents.

Clarithromycin↗

PubMed1994

Inhibitory and bactericidal activities of levofloxacin against Mycobacterium tuberculosis in vitro and in human macrophages.

Levofloxacin exhibited twofold greater inhibitory and bactericidal activities than ofloxacin against either extracellular or intracellular tubercle bacilli. The activities of both drugs against extracellular and intracellular bacteria were about the same, despite the accumulation of the drugs in macrophages at a level four- to fivefold greater than that in the extracellular medium. The activities of both drugs against intracellular bacteria were largely associated with the short, 2-h pulsed exposures of the infected macrophages to the concentrations which correspond to those attainable in blood during the period of the maximum concentration of drug in serum.

Colony Count, Microbial↗

PubMed1994

Quantitative measurement of drug susceptibility of Mycobacterium tuberculosis for monitoring chemotherapy response.

Changes in the degree of drug susceptibility of the bacterial population in tuberculosis patients during chemotherapy can be monitored by testing Mycobacterium tuberculosis isolates against various concentrations of drugs in 7H12 broth to determine radiometrically the minimal inhibitory concentration (MIC). The MICs were determined for multiple cultures isolated from 13 patients, of whom nine responded to chemotherapy within a few months, and four failed to respond at all. No changes in MICs were observed with the isolates obtained from the patients with a favorable response to chemotherapy. Determination of the MICs indicated that the failure in two cases out of four was associated with a steady increase in the degree of resistance of the patients' bacterial population. In two other failure cases, lack of change in the MIC values for multiple cultures suggests that the failure was associated with factors other than drug resistance. During the period of chemotherapy, quantitation of the degree of drug susceptibility in terms of broth-determined MICs may help to identify promptly those patients whose bacterial population is becoming resistant to the administered antimicrobial agents. For those patients who do not respond to chemotherapy, lack of change in the degree of resistance of their bacterial population may alert the physician to attempt to identify causes other than drug resistance for the chemotherapy failure.

Adult↗

PubMed1993

Low serum levels of oral antimycobacterial agents in patients with disseminated Mycobacterium avium complex disease.

Twenty-seven human immunodeficiency virus-infected patients with disseminated Mycobacterium avium complex disease who were treated with oral antimycobacterial agents (clofazimine, ciprofloxacin, ethambutol, and rifampin) were studied to evaluate the usefulness of monitoring serum drug concentrations and testing in vitro susceptibility of M. avium complex (MAC) isolates. Twenty patients tolerated treatment with three or four antimycobacterial agents for at least 8 weeks; mycobacteremia was eradicated in 7 (35%). The in vitro susceptibilities of MAC isolates to antimycobacterial agents were similar for these 7 and for the 13 who did not respond to antimycobacterial treatment. Serum drug levels were below the expected range in 6 of the 7 whose mycobacteremia was cleared and in 9 of the 13 nonresponders (P = .41). These low serum concentrations of antimycobacterial drugs may be due to impaired drug absorption in patients with AIDS and disseminated MAC disease.

AIDS-Related Opportunistic Infections↗

PubMed1993

MICs and MBCs of clarithromycin against Mycobacterium avium within human macrophages.

The inhibitory and bactericidal activities of clarithromycin were determined quantitatively against the intracellular populations of five Mycobacterium avium strains growing in monocyte-derived human macrophages. The MICs were 1.0 microgram/ml, and the MBCs ranged from 16.0 to 64.0 micrograms/ml; these values were similar to the MICs and MBCs found in broth cultures at pH 7.4 and were substantially lower than those found in broth cultures at pHs 6.8 and 5.0. Since the intracellular environment has a neutral or even an acidic pH, relatively low MICs and MBCs found in macrophage cultures can be associated with the fact that the drug concentrations in macrophages are substantially higher than those in the medium in which these cells are cultivated. Pretreatment of the macrophages 2 days prior to infection decreased the MICs twofold in comparison with results of experiments in which the drug was added to already infected macrophages.

Clarithromycin↗

PubMed1993

Mycobacterium avium strains resistant to clarithromycin and azithromycin.

Mycobacterium avium strains susceptible to clarithromycin and azithromycin contain mutants resistant to these macrolides with a frequency of 1.1 x 10(-10) to 1.2 x 10(-6). Cross-resistance between clarithromycin and azithromycin was demonstrated with mutants selected in the laboratory as well as with resistant strains isolated from patients. The susceptibility-resistance patterns of the macrolide-resistant strains with drugs other than macrolides were the same as those of the original susceptible strains. The emergence of clarithromycin resistance in patients was a result of multiplication of the preexisting resistant mutants that survived the elimination of bacteria during the initial period of treatment and was an exclusive cause of the relapse of bacteremia.

Azithromycin↗

PubMed1993

Inhibition of intracellular growth of Mycobacterium avium by one pulsed exposure of infected macrophages to clarithromycin.

A single 2-h pulsed exposure of either human monocyte-derived macrophages or J774 cells infected with Mycobacterium avium to clarithromycin at 3.0 micrograms/ml completely inhibited the intracellular bacterial growth during the first four days of observation, and then only a slight increase in the number of CFU per milliliter took place between the fourth and seventh days. These data suggest that in vivo the intracellular bacteria can be effectively inhibited after a short period when the concentration of the drug in blood reaches its maximum. On the basis of these data, the assumptions that the elimination of bacteremia observed in clarithromycin clinical trials is a result of the activity of the drug not only against bacteria in blood but in macrophages as well and that the peak concentration attainable in blood is essential for these effects can be made.

Cells, Cultured↗

PubMed1992

Pyrazinamide sterilizing activity in vitro against semidormant Mycobacterium tuberculosis bacterial populations.

Previously, we reported that pyrazinamide has very poor bactericidal activity against M. tuberculosis growing in broth at pH 5.6. In the present study, cultivation at pH 4.8 to 5.0 in 7H12 broth prevented an increase in the number of viable bacteria, but the cultures remained metabolically active. The presence of 50 micrograms/ml pyrazinamide in semidormant cultures led to a sharp decline in the number of viable bacteria, by more than 1,000-fold. This unfavorable environment probably made the bacilli especially vulnerable to pyrazinamide, whose mode of action remains unclear. To distinguish this effect of pyrazinamide on the semidormant bacteria from its mostly bacteriostatic activity against actively multiplying bacteria, we suggest interpreting the in vitro effect as "sterilizing."

Culture Media↗

PubMed1991

Collaborative feasibility study of a biphasic system (Roche Septi-Chek AFB) for rapid detection and isolation of mycobacteria.

A study to delineate the feasibility of a biphasic-culture approach for detection and isolation of mycobacteria from clinical specimens except blood was conducted in four medical centers. The biphasic system (Septi-Chek AFB, Roche Diagnostic Systems, Nutley, N.J.) was compared with conventional mycobacterial isolation media and the BACTEC system. Septi-Chek AFB showed the highest degree of mycobacterial recovery. In addition, Septi-Chek AFB consistently shortened the time required for recovery of mycobacteria from clinical specimens and supported the growth of small inoculum numbers of stock cultures of 14 mycobacterial species. The study indicates the feasibility and potential advantages of the biphasic approach for detection and isolation of mycobacteria.

Bacteriological Techniques↗

PubMed1989

Comparison of bactericidal activities of streptomycin, amikacin, kanamycin, and capreomycin against Mycobacterium avium and M. tuberculosis.

The bactericidal activities of four injectable antituberculosis drugs, streptomycin, amikacin, kanamycin, and capreomycin, against Mycobacterium avium and M. tuberculosis were tested. All four drugs were highly bactericidal against M. tuberculosis, with low MBC/MIC ratios and MBCs significantly lower than the maximum achievalbe concentrations in serum (Cmax). In contrast, all four drugs had very low bactericidal activities against M. avium: the broth-determined MBCs were significantly higher than the Cmax. On a basis of comparisons with the broth-determined MICs found for susceptible M. tuberculosis strains and with the Cmax, about one-third of 100 M. avium strains tested can be tentatively considered as susceptible to three aminoglycosides (streptomycin, amikacin, and kanamycin) but not to capreomycin. In regard to the MBCs and MICs, the three aminoglycosides tested have about identical potentials as drugs of choice in combination with other drugs for chemotherapy of M. avium disease. The low bactericidal activities of these drugs against M. avium in vitro do not exclude their therapeutic usefulness, because they may produce a synergistic effect in combination with other drugs. Such an option is especially promising for patients whose isolates can be considered susceptible on the basis of the MIC. We found no differences in susceptibility to the four drugs tested for M. avium strains (identified by Gen-Probe) isolated from 50 patients with and 50 patients without acquired immune deficiency syndrome.

Acquired Immunodeficiency Syndrome↗