Institute of Infectious Diseases and Public Health, University of Ancona, Italy
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Abstract |
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Introduction |
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Chemoprophylaxis is being used increasingly to prevent a growing number of opportunistic infections that occur in immunocompromised patients. Multiple opportunistic pathogen prophylaxis (MOPP) may result in reduced toxicity, fewer drug interactions and better patient compliance. Consequently, several studies have been focused on the antimicrobial spectrum of clinically used drugs.1,4
Co-trimoxazole is the most widely used prophylactic and therapeutic combination against P. carinii pneumonia. Drugs which inhibit folic acid synthesis have long been a mainstay in the treatment of P. carinii related infections. The dihydrofolate reductase inhibitors (DHFRs) such as pyrimethamine and trimethoprim, only have a static effect on parasite growth and frequently cause adverse effects in AIDS patients. The antifolate combination of DHFR and sulphonamide or sulphone acts synergically against P. carinii. Recent investigations have demonstrated that sulphamethoxazole accounts almost entirely for the anti-Pneumocystis activity of co-trimoxazole.5,6
A number of other single drugs and combinations of drugs are being tested in vitro and in vivo. Rifabutin has been shown to be clinically or experimentally effective against Mycobacterium spp. and Toxoplasma gondii, and it is being used extensively for prophylaxis against Mycobacterium avium complex (MAC) infections in immunocompromised HIV-positive patients.3,7 Albendazole, an agent with an important role in the treatment of cestode infections such as echinococcosis and cysticercosis, is now being used in human AIDS patients for treatment of intestinal microsporidiosis with varying results.8,9 The possible use of rifabutin and albendazole for other opportunistic infections deserves further study, although recent investigations provide evidence that both rifabutin and albendazole exhibited in-vivo activity against P. carinii in several rat models.3,10
Several studies have been performed to investigate synergy between various drugs. Recent reports provide evidence that other combinations besides co-trimoxazole have increased activity against P. carinii in vitro and in the rat model: macrolides and sulphamethoxazole, clindamycin and primaquine, atovaquone and DHFRs or rifabutin.11,12,13
In the present study we investigated the in-vitro activity of rifabutin and albendazole singly and in combination with other clinically used antimicrobial agents against P. carinii and compared their activity with that of co-trimoxazole.
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Materials and methods |
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These procedures have been described in detail previously.6Briefly four clinical isolates of P. carinii were obtained from bronchoalveolar lavages of four distinct immunocompromised patients who had not received prior anti-P. carinii therapy. Specimens were homogenized in an equal volume of phosphate-buffered saline. The homogenate was centrifuged (1800g for 15 min) and the pellet was resuspended in physiological saline. Contaminating bacteria were partially eliminated by four washes in 10 mL of physiological saline followed by incubation in phosphate-buffered saline containing ampicillin (2000 mg/L) and streptomycin (2000 mg/L), for 4 h at 37°C. Organisms were pelleted by centrifugation (1800g for 20 min), and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Bio-Whittaker, Walkersville, MD, USA), counted by methenamine silver stain to detect cysts and with Giemsa stain for counting of P. carinii nuclei prior to use for culture. The final inoculum was 103104 P. carinii organisms/mL.
The human lung epithelial cell line A-549 was maintained in 96-well tissue culture plates. Culture was initiated by adding 0.1 mL of inoculum to an adherent layer of 5070% confluent A-549 cells. After incubation for 4 h at 37°C in 5% CO2 to allow attachment and penetration of sporozoites, the monolayers were washed with DMEM to remove non-invasive sporozoites, residual cysts and nonadherent epithelial cells, and 0.2 mL of new growth medium with or without antimicrobial agents was added. Infected cell cultures were kept at 37°C in 5% CO2 throughout the study.
Drugs
All compounds were purchased commercially (Sigma-Aldrich, Milan, Italy), with the exception of clarithromycin (Abbott, Rome, Italy) and rifabutin (Pharmacia & Upjohn, Milan, Italy). Trimethoprim, pyrimethamine, clarithromycin and rifabutin were dissolved in methanol/acetone (1/1) at a concentration of 1 mg/mL. Sulphamethoxazole, albendazole and etoposide were dissolved in dimethylsulphoxide at 1 mg/mL. Minocycline was dissolved in distilled water at a concentration of 1 mg/mL. Solutions of drugs were made fresh on the day of assay or stored at 80°C in the dark for short periods.
Susceptibility testing
Serial dilutions of each drug were prepared in DMEM. All drugs were tested at concentrations close to those that could be achieved clinically. Albendazole and rifabutin were tested at concentrations of 0.5, 1, 2 and 4 mg/L. Clarithromycin, minocycline and etoposide were each tested at concentrations of 1, 2 and 4 mg/L. Pyrimethamine was tested at concentrations of 0.1, 0.2 and 0.4 mg/L. Co-trimoxazole used as a reference drug combination was tested at concentrations of 0.8, 1.6 and 3.2 mg/L, and 4, 8 and 16 mg/L of the two components, respectively. Preliminary experiments indicated that the final concentrations of methanol, acetone and dimethylsulphoxide used in the dilution of drugs did not inhibit the growth of P. carinii. In experiments to test drug interactions, the four above-mentioned concentrations of albendazole and rifabutin were added to the three concentrations of each other agent. Antibiotic-free plates were used as controls in the study. Experiments were performed in triplicate.
P. carinii were added at a concentration of 102103 organisms/well. The monolayers were incubated at 37°C in 5% CO2 atmosphere. After 72 h the supernatant was withdrawn from each triplicate well and P. carinii trophozoites and cysts counted following Giemsa and methenamine silver staining. Viability assays were performed in conjunction with microscopic enumeration of the organisms, by using the dual-fluorescence staining method.14
Analysis of results
The activity of each agent and combination was evaluated by parasite count from plates with antimicrobial-supplemented medium compared with parasite count from control plates without drugs. The average number of P. carinii parasites/mL was calculated by counting 50 oil immersion fields (x1000 magnification) of each of three slides.
The 50% and 90% inhibitory concentrations (IC50 and IC90, respectively) of each drug were defined as the concentrations required to produce a 50% and 90% reduction in the mean cyst or trophozoite counts, respectively, compared with controls without drug after 72 h incubation in the presence of drugs.
The activity of each compound was also expressed by calculating the ratio of the cyst and trophozoite numbers in cultures containing albendazole and rifabutin at a concentration of 4 mg/L, singly or in combination with other agents, to the cyst and trophozoite number in control cultures after 72 h incubation.
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Results |
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Albendazole and rifabutin had different effects (Table I). Albendazole tested alone was shown to be more effective than rifabutin, with a higher activity against trophozoites than cysts. Albendazole suppressed the growth of cysts and trophozoites by >50% at 4 mg/L (IC50 4 mg/L). Rifabutin, at the same concentration, showed less potency against either cysts or trophozoites producing about 40% reduction in the mean cyst and nucleus counts (IC50 >4 mg/L). Neither albendazole nor rifabutin achieved a 90% inhibitory effect (IC90 >4 mg/L).
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The evaluation of the ratios of the peak organism counts in drug-treated cultures to the peak organism counts in control cultures, showed that rifabutin at a concentration of 4 mg/L showed the lowest activity against either cysts or trophozoites (peak ratio, 0.60 and 0.58, respectively). Rifabutin (4 mg/L) combined with clarithromycin (4 mg/L) and etoposide (4 mg/L) showed an in-vitro anti-P.carinii activity (cyst peak ratio, 0.24 and 0.27, and trophozoite peak ratio, 0.20 and 0.23, respectively) slightly lower than co-trimoxazole (cyst peak ratio, 0.08; trophozoite peak ratio, 0.06). Albendazole tested alone at a concentration of 4 mg/L also showed weak activity (cyst peak ratio, 0.43; trophozoite peak ratio, 0.40). Albendazole (4 mg/L) upon combination with etoposide (4 mg/L) exhibited an in-vitro anti-P. carinii activity (cyst peak ratio, 0.14; trophozoite peak ratio, 0.10), similar to co-trimoxazole. These results are summarized in Table I.
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Discussion |
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Albendazole, a benzimidazole developed more than 20 years ago, is a compound that
disrupts
microtubule function by blocking polymerization of the - and ß-tubulin subunits.10 It is thought to act by binding to parasite tubulins,
inhibiting their
polymerization and impairing glucose uptake.17 It has
proved
useful in treating echinococcosis, cysticercosis and strongyloidosis. Albendazole has also been
shown to
be effective in the treatment of infections caused by the protozoans Enterocytozoon bieneusi, Septata intestinalis and Giardia intestinalis. In addition, single doses of
albendazole
have been shown to achieve cure rates of >90% against Enterobius vermicularis, Ascaris lumbricoides, hookworm and cutaneous larva migrans.17 Recently, it was evaluated for its effect on P. carinii in vitro and in
vivo and was found to be inhibitory.10,18 In our study, criteria for synergy were not defined and we did not
calculate values
such as the fractional inhibitory concentration index; thus, our findings indicate only the presence
of
additive anti-P. carinii activity when albendazole was combined with etoposide,
clarithromycin,
minocycline or pyrimethamine. The different biochemical pathways can explain the lack of
synergy
between albendazole and the other antimicrobial agents.
Rifabutin is a spiro-piperidyl-rifamycin derived from rifamycin-S. It is structurally related to and shares many properties of rifampin.19 Rifabutin has a potential role to play in a MOPP regimen because it is active against mycobacteria, Gram-positive cocci, Haemophilus influenzae, Legionella pneumophila, Helicobacter pylori, Campylobacter jejuni, Chlamydia trachomatis and Toxoplasma gondii.7 The antimicrobial activity of the rifamycins is produced by inhibition of DNA-dependent RNA polymerase and subsequent initiation of transcription. This results in inhibition of protein synthesis.19 It is currently approved in the United States, Canada and many other countries for use as prophylaxis for Mycobacterium avium- complex infection in HIV-infected patients with low CD4 cell counts. Rifabutin was shown to be highly protective against development of cryptosporidiosis in immunosuppressed HIV-infected patients.20 Recently, it was found to be highly effective in a mouse model of toxoplasmosis.21 When administered alone, rifabutin showed weak efficacy in protecting immunosuppressed rats and mice against P. carinii related pneumonia.3,13,22 In our experiments rifabutin was found to be moderately active against P. carinii, but its activity was highly enhanced upon combination with clarithromycin. Taken together, these data are in agreement with previous in-vivo investigations, in which rifabutin showed synergic activity with other clinically used agents, although these combinations were not as effective as co-trimoxazole.3 The mechanism of this synergic effect between rifabutin and clarithromycin appears to be complex. Several anti-mycobacterial in-vitro studies have shown that the effects of this combination are highly variable and dependent on the strain tested.19,23 Interestingly, studies of pharmacokinetic interaction of clarithromycin and rifabutin showed that the macrolide, by inhibiting the cytochrome P-450 pathway, reduces the metabolism of rifabutin, thus resulting in increased serum and tissue concentrations.24
Finally, in our study only the combination of co-trimoxazole suppressed the growth of cysts and trophozoites by >90%. Neither albendazole nor rifabutin, singly or in combination with other agents, achieved a 90% inhibitory effect, with the exception of albendazole (4 mg/L) associated with etoposide (4 mg/L) which produced a 90.1% reduction in trophozoite counts.
Patients with advanced HIV infection have overlapping, superimposed risks for developing protozoal, fungal, viral, bacterial and mycobacterial infections.7 The concept of MOPP is based on the premise that the parsimonious use of agents that protect against several classes of opportunistic pathogens that cause infections, will result in better efficacy, enhanced patient compliance, reduced toxicity, fewer drug interactions and lower cost. Our data showed a good anti-Pneumocystis activity of albendazole and rifabutin, used alone or in combination. However, despite this fact and the attractiveness of the concept of MOPP, there are still some significant obstacles to its implementation. The spectrum of opportunistic pathogens in immunocompromised patients is wide, and the variety of mechanisms responsible for their inhibition may necessitate the use of an array of antimicrobial agents with different modes of antimicrobial activity. In addition multidrug prophylaxis may be associated with risks of synergic or complementary toxicity and drug interaction. Clinical trials have been planned but have proved to be complex. The results of such trials would probably not be available for many years. Nevertheless albendazole and rifabutin deserve serious consideration, since these drugs, used alone or in combination, are active against many pathogens that infect individuals with HIV disease.
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Notes |
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References |
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Received 21 October 1998; returned 13 April 1999; revised 28 April 1999; accepted 29 July 1999