Marilyn S. Bartlett

Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in AIDS patients

Background:Failures of prophylaxis against Pneumocystis carinii pneumonia (PCP) in AIDS patients do occur, but no evidence for drug resistance has yet been presented. Objective:To determine whether mutations in the sulfa and sulfone drug target are associated with failure of prophylaxis using a sulfa-containing agent. Methods:Portions of the gene for P. carinii dihydropteroate synthase (DHPS), the sulfa and sulfone target, from 27 patients (20 of whom had AIDS) diagnosed with PCP between 1976 and 1997 were amplified using polymerase chain reaction and sequenced. Seven of the 27 patients (all of whom had AIDS) were receiving sulfa or sulfone drugs as prophylaxis for PCP. Results:Mutations were found at only two amino-acid positions and were significantly more common in patients who received sulfa/sulfone prophylaxis. Mutations were observed in five (71%) out of seven isolates from AIDS patients receiving sulfa/sulfone as prophylaxis compared with only two (15%) out of 13 specimens from AIDS patients who did not (P = 0.022). No mutations were seen in isolates from seven non-HIV-infected patients, none of whom were on prophylaxis. Mutations were only observed in specimens obtained in 1995–1997. Conclusions:Mutations in two amino-acid positions were significantly more common in AIDS patients with PCP who failed sulfa/sulfone prophylaxis. These amino acids appeared to be directly involved in both substrate and sulfa binding, based on homology to the Escherichia coli DHPS crystal structure. Thus, the results were consistent with the possibility that mutations in the P. carinii DHPS are responsible for some of the failures of sulfa/sulfone prophylaxis in AIDS patients.

Sequence Polymorphisms in the Pneumocystis carinii Cytochrome b Gene and Their Association with Atovaquone Prophylaxis Failure

Atovaquone (Mepron, 566c80) is an effective agent against Pneumocystis carinii, which probably acts by binding to cytochrome b and inhibiting electron transport. To assess the possibility that atovaquone resistance might be developing, the genes for the cytochrome b from P. carinii sp. f. carinii and P. carinii sp. f. hominis were partially sequenced. Eight of 10 patient isolates had cytochrome b genes with the same amino acid sequence. The P. carinii cytochrome b genes from 2 of 4 patients who had atovaquone prophylaxis failure contained mutations resulting in amino acid changes in one of the ubiquinone (coenzyme Q) binding sites (Qo). These mutations are homologous to mutations in other microorganisms that confer resistance to similar inhibitors. Variations in the sequence of the P. carinii cytochrome b gene suggest but do not prove the development of drug resistance.

The challenge of Pneumocystis carinii culture.

ABSTRACT. Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell‐free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.

Optimal Susceptibility Testing Conditions for Detection of Azole Resistance in Aspergillus spp.: NCCLS Collaborative Evaluation

ABSTRACT The most important role of susceptibility testing is to identify potentially resistant isolates for the agent being evaluated. Standard testing guidelines recently have been proposed for antifungal susceptibility testing of filamentous fungi (molds). This collaborative (eight centers) study evaluated further newly proposed guidelines (NCCLS, proposed standard M38-P, 1998) and other testing conditions for antifungal susceptibility testing of Aspergillus spp. to itraconazole and three new triazoles, posaconazole (SCH56592), ravuconazole (BMS-207147), and voriconazole. MICs of itraconazole, posaconazole, ravuconazole, and voriconazole for 15 selected isolates of three species of Aspergillus (A. fumigatus, A. flavus, and A. terreus) with well documented in vitro, clinical, or animal data were determined in each center by using four medium formulations (standard RPMI-1640 [RPMI], RPMI with 2% dextrose, antibiotic medium 3 [M3], and M3 with 2% dextrose) and two criteria of MIC determination (complete [MIC-0s] and prominent [MIC-2s] growth inhibition) at 24, 48, and 72 h. The highest reproducibility (92 to 99%) was seen with the standard RPMI and M3 media. Moreover, the distinction between itraconazole-resistant (MICs of >8 μg/ml for clinically resistant strains) and -susceptible (MICs of 0.03 to 1 μg/ml) isolates, as well as between a voriconazole-resistant laboratory mutant and other isolates (voriconazole MICs of 2 to >8 versus 0.12 to 2 μg/ml), was more consistently evident with the standard RPMI medium and when MIC-0s were determined at 48 h. These results provide further refinement of the testing guidelines for susceptibility testing ofAspergillus spp. and warrant consideration for inclusion in the future NCCLS document M38-A.

The β-tubulin gene from rat and human isolates of Pneumocystis carinii

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long‐term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short‐term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the β‐tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single β‐tubulin gene from a rat P. carinii isolate. There was 89‐91% identity at the amino acid level to β‐tubulins from filamentous fungi, but only 79‐82% identity to yeast and protozoal β‐tubulins. Also, eight introns were distributed throughout the P. cariniiβ‐tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. cariniiβ‐tubulin. The polymerase chain reaction was used to amplify a segment of P. cariniiβ‐tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the ammo‐acid level.

Activity of clindamycin with primaquine against Pneumocystis carinii in vitro and in vivo.

The combination of primaquine with clindamycin is effective in both in vitro and in vivo models of Pneumocystis infection. Primaquine alone at concentrations from 10 to 300 micrograms/ml reduced the numbers of organisms in cultures to less than 7% of control. Significant inhibition was observed down to 0.1 microgram/ml. Clindamycin at 5 micrograms/ml was ineffective alone. Combinations of clindamycin and primaquine in culture at various concentrations were effective, but there was no evidence of true synergy. In rats with established Pneumocystis pneumonia, clindamycin alone at 5 or 225 mg/kg was ineffective. Primaquine alone at 0.5 or 2 mg/kg did not significantly affect the numbers of organisms remaining. The combination of 0.5 mg of primaquine per kg and 225 mg of clindamycin per kg was effective for therapy, lowering the numbers of organisms in the lungs by about 90%. The combination of 2 mg of primaquine per kg and 225 mg of clindamycin per kg was more effective, lowering the numbers of organisms by almost 98%. In the in vivo prophylaxis model, primaquine at 0.1 or 0.2 mg/kg did not prevent the development of Pneumocystis pneumonia in immune-suppressed rats. Clindamycin at 50 mg/kg had a modest effect alone, but at 5 mg/kg all animals became heavily infected. At 0.5 mg/kg, primaquine alone reduced the severity of infection, but seven of eight rats were still infected. In contrast, the combination of 5 mg of clindamycin per kg and 0.5 mg of primaquine per kg prevented infection in 8 of 10 rats; 2 rats had minimal infection. These studies suggest that the combination of clindamycin and primaquine should be tested in therapy or prophylaxis of Pneumocystis infections in humans. Images

Correlation of Organism Burden and Alveolar Macrophage Counts during Infection with Pneumocystis carinii and Recovery

ABSTRACT Changes in the number of alveolar macrophages were correlated with organism burden during Pneumocystis carinii infection. The lungs of healthy, dexamethasone-treated, and dexamethasone-treated and P. carinii-infected rats were lavaged with phosphate-buffered saline. Counting of alveolar macrophages in the lavage fluids revealed that P. carinii infection caused a 58% decrease in the number of alveolar macrophages and that higher P. carinii organism burdens caused a more rapid decrease in alveolar macrophage number. As a control, healthy rats were challenged with the same number of organisms as that normally used to generate P. carinii infections in dexamethasone-treated rats. Thirteen days after challenge, these rats had a profound (54%) increase in alveolar macrophage number in response to the challenge, while the number of alveolar macrophages in immunosuppressed and P. carinii-infected rats had decreased significantly by this time point. These experiments created the first animal model to mimic human pneumocystis pneumonia in alveolar macrophage number alterations. Reduction of P. carinii organism numbers by treatment of rats with trimethoprim and sulfamethoxazole brought a slow rebound in alveolar macrophage number, while recovery from P. carinii infection by cessation of immunosuppression brought a rapid rebound in alveolar macrophage number. These results suggest that both the immune state of the host and P. carinii burden affect alveolar macrophage number.

Antiparasitic activities and toxicities of individual enantiomers of the 8-aminoquinoline 8-[(4-amino-1-methylbutyl)amino]-6-methoxy-4-methyl-5-[3,4- dichlorophenoxy]quinoline succinate

ABSTRACT 8-Aminoquinolines are an important class of antiparasitic agents, with broad utility and excellent efficacy, but also limitations due to hematological toxicities, primarily methemoglobinemia and hemolysis. One representative from this class, (±)-8-[(4-amino-1-methylbutyl)amino]-6-methoxy-4-methyl-5-[3,4-dichlorophenoxy]quinoline succinate (NPC1161C), proved extremely efficacious in animal models of malaria and pneumocystis pneumonia. This racemic mixture was separated into its component enantiomers by chemical and chromatographic means. The enantiomers were evaluated for antiparasitic activity in murine models of Plasmodium berghei, Pneumocystis carinii, and Leishmania donovani infection, as well as the propensity to elicit hematotoxicity in dogs. The (−)-enantiomer NPC1161B was found to be more active (by severalfold, depending on the dosing regimen) than the (+)-enantiomer NPC1161A in all of these murine models. In addition, the (−) enantiomer showed markedly reduced general toxicity in mice and reduced hematotoxicity in the dog model of methemoglobinemia. It is concluded that the configuration at the asymmetric center in the 8-amino side chain differentially affects efficacy and toxicity profiles and thus may be an important determinant of the “therapeutic window” for compounds in this class.

Activity of lipid-soluble inhibitors of dihydrofolate reductase against Pneumocystis carinii in culture and in a rat model of infection.

Trimetrexate and BW301U (piritrexim isethionate), lipid-soluble inhibitors of dihydrofolate reductase, are potent inhibitors of the growth of Pneumocystis carinii in culture with WI-38 cells. Inhibition was observed with 0.1 microgram of trimetrexate or BW301U per ml. Trimethoprim is ineffective at 100 micrograms/ml in this culture system. Both trimetrexate and BW301U were effective as prophylactic agents against P. carinii pneumonia in rats; trimetrexate at 7.5 mg/kg protected 9 of 10 rats, and BW301U at 5 mg/kg protected 4 of 10.

Pneumocystis carinii is resistant to imidazole antifungal agents.

Because Pneumocystis carinii is closely related to fungi, drugs useful for treating mycoses have been considered for use in the treatment of P. carinii pneumonia. Six antifungal imidazole drugs were tested for their activities against P. carinii in a short-term culture screen and in animals. None of the imidazoles tested was effective in inoculated infected rats, and only miconazole showed slight effects in culture at the high concentration of 10 micrograms/ml. Analysis of cell membranes from culture-grown P. carinii showed that ergosterol, the target for this class of antifungal agents, was absent, so that the lack of effect of these agents is rational.