bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified Fixed-Ratio Isobologram Method for Studying In Vitro Interactions between Atovaquone and Proguanil or Dihydroartemisinin against Drug-Resistant Strains of
Plasmodium falciparum
Authors: Fivelman Quinton L. , Adagu Ipemida S. , Warhurst David C.
Published: October 2004

A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described. This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin. The interaction between atovaquone and proguanil was synergistic against atovaquone-sensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure. While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested. The relevance of in vitro interactions to in vivo treatment is discussed.
Emergence of Multidrug-Resistant
Salmonella enterica
Serovar Typhi in Korea
Authors: Lee Kyungwon , Yong Dongeun , Yum Jong Hwa , Lim Young Sik , Kim Hyun Sook , Lee Bok Kwon , Chong Yunsop
Published: October 2004

A chloramphenicol-resistant strain of
Salmonella enterica
serovar Typhi was first noted in Korea in 1992, when a resistant isolate was detected in a returned traveler. Continued isolation of multidrug-resistant (MDR) strains thereafter in other settings prompted a retrospective analysis of laboratory records and phenotypic and genotypic analyses of 12 chloramphenicol-resistant isolates. Among these, one isolate was resistant only to chloramphenicol, and the other isolates were also resistant to ampicillin and co-trimoxazole. MDR was transferred by conjugation from 9 of the 11 isolates. PCR showed that all isolates had an incompatible group HI1 plasmid, and
oriT
was detected in 10 isolates, which included strains with an unsuccessful transfer of resistance. All of the ampicillin-resistant isolates had a β-lactamase band of pI 5.4 and
bla
TEM
alleles. A PCR amplicon from an isolate showed that the sequences were identical to those of
bla
TEM-1
, suggesting that all isolates had a TEM-1 β-lactamase. All isolates had class 1 integrons: 10 isolates had integrons of ca. 1.2 kb with
dhfr7
gene cassettes, and 1 isolate had an integron of ca. 2.3 kb with
aacA4
and
bla
OXA-1
-like gene cassettes. The pulsed-field gel electrophoresis patterns of 7 of 11 MDR isolates were identical and indistinguishable from those reported for isolates in India and Indonesia. In conclusion, some of the MDR strains in Korea are related to those in other Asian countries. Susceptibility testing became necessary for selection of antimicrobial agents for the optimal treatment of patients with the emergence of MDR
Salmonella
serovar Typhi in Korea.
Four Variants of the
Citrobacter freundii
AmpC-Type Cephalosporinases, Including Novel Enzymes CMY-14 and CMY-15, in a
Proteus mirabilis
Clone Widespread in Poland
Authors: Literacka Elżbieta , Empel Joanna , Baraniak Anna , Sadowy Ewa , Hryniewicz Waleria , Gniadkowski Marek
Published: October 2004

Twenty-nine
Proteus mirabilis
isolates from 17 Polish hospitals were analyzed. The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to β-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers. Indeed, β-lactamases with a pI of ∼9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants. The enzymes were of
Citrobacter freundii
origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones. The
bla
CMY
genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of ∼10 kb. In the CMY-12- and -15-producing isolates, an additional fragment of ∼4.5 kb hybridized with the
bla
CMY
probe as well, which could have arisen from a duplication event during the evolution of the genes. In all of the isolates, the IS
Ecp1
mobile element, which most probably is involved in mobilization of the
C. freundii ampC
gene, was placed at the same distance from the 5′ ends of the
bla
CMY
genes, and sequences located between them were identical in isolates carrying each of the four genes. These data suggested that a single chromosome-to-chromosome transfer of the
ampC
gene from
C. freundii
to
P. mirabilis
could have initiated the spread and evolution of the AmpC-producing
P. mirabilis
in Poland. The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the
P. mirabilis
isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates.
Effects of Amoxicillin Subinhibitory Concentrations on the Cross-Protection Developed by Pneumococcal Antibodies in Mouse Sepsis Caused by an Amoxicillin-Resistant Serotype 6BStreptococcus pneumoniaeStrain
Authors: Tarragó D. , Aguilar L. , Giménez M. J. , Fenoll A. , Casal J.
Published: October 2004

A model of mouse sepsis caused by a serotype 6BStreptococcus pneumoniaestrain (amoxicillin MIC of 8 μg/ml) was developed to investigate the therapeutic effect of an amoxicillin dose (3.12 mg/kg of body weight three times daily for 48 h) producing, over the whole treatment period, subinhibitory concentrations in serum (peak concentration [Cmax]: 6.1 μg/ml) in animals that prior to infection had been passively immunized with a 6B or 23F hyperimmune serum (obtained by immunization with a whole-cell heat-inactivated inoculum and diluted to produce no protective effect by itself). Mortality in nonimmunized animals treated with antibiotic (3.12 mg/kg) was 90%, and mortality in animals immunized but not treated with the antibiotic was 100%. Antibiotic treatment in immunized animals produced mortality rates ≤20% when the hyperimmune serum was used, thus showing cross-protection and synergism (defined as the situation in which there is no response to the single agents [no differences versus placebo] while the combination exhibits significant activity) with subinhibitory concentrations of the antibiotic. The presence of antipneumococcal antibodies allowed antibiotic efficacy with negligible values of pharmacodynamic parameters (Cmax/MIC ratio of <1 and thus a null value for the time that serum levels exceed the MIC). This in vivo synergism offers a potential therapeutic strategy against resistant strains.
Nevirapine Significantly Reduces the Levels of Racemic Methadone and (R)-Methadone in Human Immunodeficiency Virus-Infected Patients
Authors: Stocker Hartmut , Kruse Guido , Kreckel Peter , Herzmann Christian , Arastéh Keikawus , Claus Jörg , Jessen Heiko , Cordes Christiane , Hintsche Bettina , Schlote Frank , Schneider Lothar , Kurowski Michael
Published: October 2004

Methadone is metabolized by various isoforms of the cytochrome P450 family, which can be induced by many drugs, including nevirapine. The objective of the present study was to determine the effects of coadministration of nevirapine and methadone on the dose-adjusted areas under the concentration-time curves (AUCs) of racemic and (R)-methadone. Twenty-five human immunodeficiency virus-infected subjects taking stable single daily doses of racemic methadone or (R)-methadone were included in this prospective, single-crossover trial. At the baseline, nevirapine was either started as part of a new regimen containing two nucleoside reverse transcriptase inhibitors (NRTIs) or added to an ongoing NRTI regimen. Patients could increase their methadone doses if withdrawal symptoms developed. Twelve-hour pharmacokinetic profiles were obtained before and 28 days after the start of nevirapine treatment. The total concentrations of methadone and its inactive metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in serum were determined by liquid chromatography-tandem mass spectrometry. Among the 20 evaluable patients, coadministration of nevirapine significantly decreased the mean dose-adjusted AUC of methadone by 41%. AUC reductions were similar for patients taking racemic methadone (37%;n= 11) and (R)-methadone (44%;n= 9). AUC changes ranged from mild increases in three patients to decreases of up to 70%. Fourteen of 20 patients required additional methadone due to withdrawal symptoms. However, the median dose increase was only 15%, which was less than that which would have been expected from the pharmacokinetic data. The AUC of EDDP increased significantly, by 35%. Methadone dose adjustments are justified when methadone is coadministered with nevirapine. Due to extensive variability, the adjustments must be tailored to the individual patient's needs.
Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity
Authors: Herget Thomas , Freitag Martina , Morbitzer Monika , Kupfer Regina , Stamminger Thomas , Marschall Manfred
Published: October 2004

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC
50
s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC
50
s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC
50
of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.
Multidrug Resistance of a Porin Deletion Mutant of
Mycobacterium smegmatis
Authors: Stephan Joachim , Mailaender Claudia , Etienne Gilles , Daffé Mamadou , Niederweis Michael
Published: October 2004

Mycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents. It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly. A mutant of
Mycobacterium smegmatis
lacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity. Deletion of the
mspA
gene caused high-level resistance of
M. smegmatis
to 256 μg of ampicillin/ml by increasing the MIC 16-fold. The permeation of cephaloridine in the
mspA
mutant was reduced ninefold, and the resistance increased eightfold. This established a clear relationship between the activity and the outer membrane permeation of cephaloridine. Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the
mspA
mutant were increased 2- to 10-fold. This is in contrast to those for
Escherichia coli
, whose sensitivity to these agents was not affected by deletion of porin genes. Uptake of the very hydrophobic steroid chenodeoxycholate by the
mspA
mutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway. The multidrug resistance of the
mspA
mutant highlights the prominent role of outer membrane permeability for the sensitivity of
M. smegmatis
to antibiotics. An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria.
Inhibition of Antibiotic Efflux in Bacteria by the Novel Multidrug Resistance Inhibitors Biricodar (VX-710) and Timcodar (VX-853)
Authors: Mullin Steve , Mani Nagraj , Grossman Trudy H.
Published: October 2004

Inhibitors of mammalian multidrug efflux, such as the plant alkaloid reserpine, are also active in potentiating antibiotic activity by inhibiting bacterial efflux. Based on this precedent, two novel mammalian multiple drug resistance inhibitors, biricodar (VX-710) and timcodar (VX-853), were evaluated for activity in a variety of bacteria. Both VX-710 and VX-853 potentiated the activity of ethidium bromide (EtBr), a model efflux substrate, against three clinically significant gram-positive pathogens:
Staphylococcus aureus
,
Enterococcus faecalis
, and
Streptococcus pneumoniae
. Similar to reserpine, VX-710 and VX-853 directly blocked EtBr efflux in
S. aureus
. Furthermore, these compounds were effective in lowering the MICs of several clinically used antibiotics, including fluoroquinolones, suggesting that VX-710 and VX-853 are representatives of a new class of bacterial efflux inhibitors with the potential for use in combination therapy.
Promoter Sequences Necessary for High-Level Expression of the Plasmid-AssociatedampCβ-Lactamase GeneblaMIR-1
Authors: Reisbig Mark D. , Hanson Nancy D.
Published: October 2004

Little is known about mechanisms involved in high-level expression of plasmid-associatedampCgenes. The sequence forblaMIR-1has been elucidated, and the gene is not inducible. Although the sequence for the promoter (prA) that drives expression ofEnterobacter cloacaechromosomalampCis present upstream ofblaMIR-1, high-level expression fromblaMIR-1is directed from a hybrid promoter (prB) located further upstream of prA. The purpose of this study was to determine the influence of each promoter onblaMIR-1expression and β-lactam resistance. RNA expression by deletion clones with both promoters was measured and compared to that by clones in which −35 and/or −10 elements of prA and/or prB were altered. Primer extension revealed two start sites forblaMIR-1transcription. Expression ofblaMIR-1in clones with both promoters was 171-fold higher than that in clones carrying only prA. In addition,blaMIR-1expression from prA increased 11-fold in the presence of the prB −10 element compared to expression driven from prA alone. Ceftazidime and cefotaxime MICs increased 42- and 64-fold, respectively, for the clone expressingblaMIR-1from both promoters compared to expression from prA alone. The upstream promoter prB ofblaMIR-1is solely responsible for high-level expression required for cefotaxime and ceftazidime resistance. These data suggest that resistance to extended-spectrum cephalosporins mediated by noninducible plasmid-associatedampCgenes requires the formation of novel promoter elements that are capable of increasingampCexpression.
Emergence and Persistence of Macrolide Resistance in Oropharyngeal Flora and Elimination of Nasal Carriage of
Staphylococcus aureus
after Therapy with Slow-Release Clarithromycin: a Randomized, Double-Blind, Placebo-Controlled Study
Authors: Berg Hans F. , Tjhie Jeroen H. T. , Scheffer Gert-Jan , Peeters Marcel F. , van Keulen Peter H. J. , Kluytmans Jan A. J. W. , Stobberingh Ellen E.
Published: October 2004

To investigate the effect of slow-release (SR) clarithromycin on colonization and the development of resistance in oropharyngeal and nasal flora, a double-blind, randomized, placebo-controlled trial was performed with 8 weeks of follow-up. A total of 296 patients with documented coronary artery disease were randomized in the preoperative outpatient clinic to receive a daily dose of SR clarithromycin (500 mg) (CL group) or placebo tablets (PB group) until the day of surgery. Nose and throat swabs were taken before the start of therapy, directly after the end of therapy, and 8 weeks later. The presence of potential pathogenic bacteria was determined, and if they were isolated, MIC testing was performed. Quantitative culture on media with and without macrolides was performed for the indigenous oropharyngeal flora. In addition, analysis of the mechanism of resistance was performed with the macrolide-resistant indigenous flora. Basic patient characteristics were comparable in the two treatment groups. The average number of tablets taken was 15 (standard deviation = 6.4). From the throat swabs,
Haemophilus parainfluenzae
was isolated and carriage was not affected in either of the treatment groups. Nasal carriage of
Staphylococcus aureus
, however, was significantly reduced in the CL group (from 35.3 to 4.3%) compared to the PB group (from 32.4 to 30.3%) (
P
< 0.0001; relative risk [RR], 7.0; 95% confidence interval [CI], 3.1 to 16.0). Resistance to clarithromycin was present significantly more frequently in
H. parainfluenzae
in the CL group after treatment (
P
= 0.007; RR, 1.6; 95% CI, 1.1 to 2.3); also, the percentage of patients with resistance to macrolides in the indigenous flora after treatment was significantly higher in the CL group (31 to 69%) (
P
< 0.0001; RR, 1.9; 95% CI, 1.4 to 2.5). This persisted for at least 8 weeks. This study shows that besides the effective elimination of nasal carriage of
S. aureus
, treatment with SR clarithromycin for approximately 2 weeks has a marked and sustained effect on the development of resistance in the oropharyngeal flora for at least 8 weeks.
Differential Maintenance of the M184V Substitution in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 by Various Nucleoside Antiretroviral Agents in Tissue Culture
Authors: Petrella Marco , Oliveira Maureen , Moisi Daniela , Detorio Mervi , Brenner Bluma G. , Wainberg Mark A.
Published: October 2004

The M184V substitution in human immunodeficiency virus type 1 reverse transcriptase (RT) is rapidly selected in tissue culture following serial passage of wild-type virus in the presence of increasing concentrations of lamivudine (3TC). M184V is also associated with several alterations of RT enzymatic function in vitro that may adversely affect viral fitness or replication capacity, which creates a potential rationale for its maintenance once it has been selected by antiviral chemotherapy. However, the relative effectiveness of nucleoside RT inhibitors that are structurally unrelated to 3TC in selecting and/or maintaining M184V has not been investigated. In the present study, we have studied the abilities of a variety of drugs, i.e., zalcitabine (ddC), didanosine (ddI), abacavir (ABC), and the novel nucleoside SPD754, in addition to 3TC, to maintain the presence of M184V in tissue culture and have shown that SPD754, ABC, and 3TC are able to preserve M184V in mixed dual infections consisting of wild-type viruses and clinical isolates which contained the M184V mutation. Moreover, M184V could also be maintained in these cultures when a subtherapeutic concentration of 3TC (i.e., 0.05 μM) was used. In contrast, neither ddI nor ddC was able to maintain M184V to the same extent as the other drugs after 10 weeks of tissue culture in mixtures of wild-type viruses and isolates containing M184V in different proportions.
Pharmacodynamics of Pulse Dosing versus Standard Dosing: In Vitro Metronidazole Activity against
Bacteroides fragilis
and
Bacteroides thetaiotaomicron
Authors: Ibrahim Khalid H. , Gunderson Brent W. , Hermsen Elizabeth D. , Hovde Laurie B. , Rotschafer John C.
Published: October 2004

Pulse dosing is a novel approach to dosing that produces escalating antibiotic levels early in the dosing interval followed by a prolonged dose-free period. Antibiotic is frontloaded by means of four sequential bolus injections, after which antibiotic levels are allowed to diminish until the next dose. This study compares standard thrice-daily dosing and pulse dosing of metronidazole against
Bacteroides
spp. in an in vitro model. Two American Type Culture Collection
Bacteroides fragilis
isolates (metronidazole MIC for each organism = 1 mg/liter) were exposed to metronidazole for 48 or 96 h. Human pharmacokinetics were simulated for an oral 500-mg dose given every 8 h (maximum concentration of drug [
C
max
] = 12 mg/liter; half-life = 8 h; area under the curve [AUC] = 294 mg · h/liter) and for pulse dosing. Pulses, each producing an increase in metronidazole concentration of 9 mg/liter, were administered at times 0, 2, 4, and 6 h of each 24-h cycle, with a targeted half-life of 8 h (AUC = 347 mg · h/liter). A metronidazole-resistant
B. fragilis
strain (metronidazole MIC = 32 mg/liter) was exposed to both dosing regimens and, additionally, to a regimen of 1,500 mg administered once daily (
C
max
= 36 mg/liter; AUC = 364 mg · h/liter). Furthermore, regimens against one
B. fragilis
isolate and one
B. thetaiotaomicron
isolate corresponding to one-fourth and one-eighth of the thrice-daily and pulse dosing regimens, mimicking peak metronidazole concentrations achieved in abscesses, were simulated in 48-h experiments (metronidazole MIC = 1 mg/liter). Time-kill curves were generated for each experiment and analyzed for bactericidal activity, defined as a bacterial burden reduction ≥ 3 log
10
CFU/ml. The results of paired (Wilcoxon matched-pair signed-rank test) and nonpaired (Mann-Whitney test) statistical analyses conducted on time to 3 log
10
kill data and area under the kill curve data from each of the thrice-daily dosing experiments versus each of the pulse dosing experiments were considered not significant for a given isolate-dosing regimen combination. The thrice-daily dosing, pulse dosing, and once-daily dosing regimens all exhibited bactericidal activity. Metronidazole administered in standard or pulse dosing fashion was highly active against both susceptible and resistant strains of
Bacteroides
spp.
Pharmacokinetics of Indinavir and Ritonavir Administered at 667 and 100 Milligrams, Respectively, Every 12 Hours Compared with Indinavir Administered at 800 Milligrams Every 8 Hours in Human Immunodeficiency Virus-Infected Patients
Authors: Rhame Frank S. , Rawlins Sandy L. , Petruschke Richard A. , Erb Tara A. , Winchell Gregory A. , Wilson Helene M. , Edelman Jonathan M. , Abramson Murray A.
Published: October 2004

Human immunodeficiency virus (HIV) patients on nucleoside or nucleotide reverse transcriptase inhibitors with HIV RNA at <1,000 copies/ml were randomized in an open-label study to administration of combined indinavir/ritonavir (IDV/RTV) at 667/100 mg every 12 h (q12h) or IDV alone at 800 mg q8h to determine the regimens' pharmacokinetics. On day 14, plasma IDV and RTV levels were determined over 24 h. Noncompartmental pharmacokinetics (minimum concentration of drug in serum [
C
min
], area under the concentration-time curve from 0 to 24 h [AUC
0-24
], and maximum concentration of drug in serum [
C
max
]) were expressed as geometric mean values with 90% confidence intervals (CI). The primary hypothesis was that the lower bound of the protocol-specified 90% CI for the geometric mean
C
min
ratio of the combination compared to IDV alone regimen would be ≥2. Twenty-seven patients were enrolled, and 24 (15 male; average age, 42 years) completed the study. The
C
min
, AUC
0-24
, and
C
max
for IDV/RTV compared to IDV alone were 1,511 versus 250 nM, 119,557 versus 77,034 nM · h, and 10,428 versus 10,407 nM, respectively. Corresponding relationships for IDV/RTV compared to IDV alone were a 6.0-fold increase in
C
min
(90% CI, 4.0, 9.3), an increase in AUC
0-24
(1.5-fold, 90% CI, 1.2, 2.0), and no increase in
C
max
. Adverse events were similar and generally mild, with no cases of nephrolithiasis. The geometric mean ratio of IDV
C
min
for IDV/RTV compared to IDV was at least 2 by a lower bound of the 90% CI, satisfying the primary hypothesis. The
C
max
was not increased, suggesting an IDV/RTV 667/100-mg toxicity profile may be similar to that of unboosted IDV.
Highly Active Anti-
Pneumocystis carinii
Compounds in a Library of Novel Piperazine-Linked Bisbenzamidines and Related Compounds
Authors: Cushion Melanie T. , Walzer Peter D. , Collins Margaret S. , Rebholz Sandra , Vanden Eynde Jean Jacques , Mayence Annie , Huang Tien L.
Published: October 2004

Trimethoprim-sulfamethoxazole and pentamidine isethionate have been used extensively for the prophylaxis and therapy of pneumonia caused by
Pneumocystis jirovecii
. Problems associated with toxicity and potential emerging resistance for both therapies necessitate the development of safe and effective analogs or new treatment strategies. In the present study, a library of 36 compounds was synthesized by using the pentamidine molecule as the parent compound modified by a 1,4-piperazinediyl moiety as the central linker to restrict conformation flexibility. The compounds were evaluated for anti-
Pneumocystis carinii
activity in a bioluminescent ATP-driven assay. Four of the compounds were highly active, with 50% inhibitory concentration (IC
50
) values of <0.01 μg/ml; four had very marked activity (IC
50
< 0.10 μg/ml); ten had marked activity (IC
50
< 1.0 μg/ml); nine had moderate activity (IC
50
< 10 μg/ml); one had slight activity (IC
50
= 34.1 μg/ml); and the remaining eight did not demonstrate activity in this assay system. The high level of activity was specifically associated with an alkyl chain length of five to six carbons attached to one of the nitrogens of the bisamidinium groups. None of the highly active compounds and only one of the very marked compounds exhibited any toxicity when evaluated in three mammalian cell lines. The strategy of substitution of 1,4-piperazine-linked bisbenzamidines produced compounds with the highest level of activity observed in the ATP assay and holds great promise for the development of efficacious anti-
P. carinii
therapy.
Comparative Pharmacokinetics of Intramuscular Artesunate and Artemether in Patients with Severe Falciparum Malaria
Authors: Hien T. T. , Davis T. M. E. , Chuong L. V. , Ilett K. F. , Sinh D. X. T. , Phu N. H. , Agus C. , Chiswell G. M. , White N. J. , Farrar J.
Published: October 2004

The first-dose pharmacokinetic properties of intramuscular (i.m.) artesunate (ARTS; 2.4 mg/kg immediately [stat], followed by 1.2 mg/kg i.m. daily) and artemether (ARM; 3.2 mg/kg i.m. stat, followed by 1.6 mg/kg i.m. daily) were compared in Vietnamese adults with severe falciparum malaria. A total of 19 patients were studied; 9 received ARTS, and 10 received ARM. ARTS was absorbed very rapidly; concentrations in plasma peaked between 1,362 and 8,388 nmol/liter (median, 5,710 nmol/liter) within 20 min of injection and then declined with a median (range) half-life (
t
1/2
) of 30 (3 to 67) min. ARTS was hydrolyzed rapidly and completely to the biologically active metabolite dihydroartemisinin (DHA). Peak DHA concentrations in plasma ranged between 1,718 and 7,080 nmol/liter (median, 3,060 nmol/liter) and declined with a
t
1/2
of 52 (26 to 69) min. In contrast, ARM was slowly and erratically absorbed. The absorption profile appeared biphasic. Maximum ARM concentrations in plasma ranged between 67 nmol/liter (a value close to the 50% inhibitory concentration for some
Plasmodium falciparum
isolates) and 1,631 nmol/liter (median, 574 nmol/liter) and occurred at a median (range) of 10 (1.5 to 24) h. There was relatively little conversion to DHA. After i.m. injection in cases of severe malaria, absorption of the water-soluble ARTS is rapid and extensive, whereas the oil-based ARM is slowly and erratically absorbed, with relatively little conversion to the more active DHA. On the basis of this pharmacological study, parenteral ARTS is preferable to ARM as an initial antimalarial therapy, particularly in the most seriously ill patients. These findings should be formally assessed by a randomized clinical trial.
Concentrations of Gemifloxacin at the Target Site in Healthy Volunteers after a Single Oral Dose
Authors: Islinger Florian , Bouw Rene , Stahl Mathias , Lackner Edith , Zeleny Petra , Brunner Martin , Müller Markus , Eichler Hans Georg , Joukhadar Christian
Published: October 2004

Free gemifloxacin concentrations in the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue were measured by means of in vivo microdialysis to characterize the ability of gemifloxacin to penetrate human soft tissues. Twelve healthy volunteers received a single oral dose of 320 mg of gemifloxacin. The mean areas under the concentration-time curves from 0 to 10 h (AUC
0-10
) were significantly higher for soft tissue than for unbound gemifloxacin in plasma (
P
< 0.05). The ratios of the mean AUC
0-10
for tissue to the AUC
0-10
for free gemifloxacin in plasma were 1.7 ± 0.7 (mean ± standard deviation) for skeletal muscle and 2.4 ± 1.0 for adipose tissue. The AUC
0-24
ratios for free gemifloxacin in tissues to the MIC at which 90% of frequently isolated bacteria are inhibited were close to or higher than 100 h. Therefore, based on pharmacokinetic and pharmacodynamic calculations, we conclude that gemifloxacin might be a useful therapeutic option for the treatment of soft tissue infections.
Complex Multiple Antibiotic and Mercury Resistance Region Derived from the r-det of NR1 (R100)
Authors: Partridge Sally R. , Hall Ruth M.
Published: October 2004

The sequence of the 45.2-kb multidrug and mercury resistance region of pRMH760, a large plasmid from a clinical isolate of
Klebsiella pneumoniae
collected in 1997 in Australia, was completed. Most of the modules found in the resistance determinant (r-det), or Tn
2670
, region of NR1 (also known as R100), isolated from a
Shigella flexneri
strain in Japan in the late 1950s, were present in pRMH760 but in a different configuration. The location was also different, with the Tn
2670
-derived region flanked by the transposition module of Tn
1696
and a mercury resistance module almost identical to one found in the plasmid pDU1358. This arrangement is consistent with a three-step process. First, the r-det was circularized via homologous recombination between the IS
1
elements and reincorporated at a new location, possibly in a different plasmid, via homologous recombination between the 5′-conserved (5′-CS) or 3′-CS of the In34 integron in the r-det and the same region of a second class 1 integron in a Tn
1696
relative. Subsequently, resolvase-mediated recombination between the
res
sites in the r-det and a second mercury resistance transposon removed one end of the Tn
1696
-like transposon and part of the second transposon. Other events occurring within the r-det-derived portion have also contributed to the formation of the pRMH760 resistance region. Tn
2
or a close relative that includes the
bla
TEM-1b
gene had moved into the Tn
21
mercury resistance module with subsequent deletion of the adjacent sequence, and all four 38-bp inverted repeats corresponding to Tn
21
family transposon termini have been interrupted by an IS
4321
-like element.
Steady-State Pharmacokinetics of a Double-Boosting Regimen of Saquinavir Soft Gel plus Lopinavir plus Minidose Ritonavir in Human Immunodeficiency Virus-Infected Adults
Authors: Ribera Esteban , Lopez Rosa M. , Diaz Marjorie , Pou Leonor , Ruiz Lidia , Falcó Vicenç , Crespo Manuel , Azuaje Carlos , Ruiz Isabel , Ocaña Imma , Clotet Bonaventura , Pahissa Albert
Published: October 2004

Management of treatment-experienced human immunodeficiency virus patients has become complex, and therapy may need to include two protease inhibitors at therapeutic doses. The objective of this study was to characterize the pharmacokinetics in serum of saquinavir (1,000 mg twice daily [b.i.d.]), lopinavir (400 mg b.i.d.), and ritonavir (100 mg b.i.d.) in a multidrug rescue therapy study and to investigate whether steady-state pharmacokinetics of lopinavir-ritonavir are affected by coadministration of saquinavir. Forty patients were included (25 given ritonavir, lopinavir, and saquinavir and 15 given ritonavir and lopinavir). The median pharmacokinetic parameters of lopinavir were as follows: area under the concentration-time curve from 0 to 12 h (AUC
0-12
), 85.1 μg/ml · h; maximum concentration of drug in serum (
C
max
), 10.0 μg/ml; trough concentration of drug in serum (
C
trough
), 7.3 μg/ml; and minimum concentration of drug in serum (
C
min
), 5.5 μg/ml. Lopinavir concentrations were similar in patients with and without saquinavir. The median pharmacokinetic parameters for saquinavir were as follows: AUC
0-12
, 22.9 μg/ml · h;
C
max
, 2.9 μg/ml;
C
trough
, 1.6 μg/ml; and
C
min
, 1.4 μg/ml. There was a strong linear correlation between lopinavir and ritonavir and between saquinavir and ritonavir concentrations in plasma. The correlation between lopinavir and saquinavir levels was weaker. We found higher saquinavir concentrations in women than in men, with no difference in lopinavir levels. Only patients with very high body weight presented lopinavir and saquinavir concentrations lower than the overall group. Ritonavir has a double-boosting function for both lopinavir and saquinavir, and in terms of pharmacokinetics, the drug doses selected seemed appropriate for combining these agents in a dual protease inhibitor-based antiretroviral regimen for patients with several prior virologic failures.
In Vivo Assessment of Drug Efficacy against
Plasmodium falciparum
Malaria: Duration of Follow-Up
Authors: Stepniewska Kasia , Taylor Walter R.J. , Mayxay Mayfong , Price Ric , Smithuis Frank , Guthmann Jean-Paul , Barnes Karen , Myint Hla Yin , Adjuik Martin , Olliaro Piero , Pukrittayakamee Sasithon , Looareesuwan Sornchai , Hien Tran Tinh , Farrar Jeremy , Nosten François , Day Nicholas P.J. , White Nicholas J.
Published: October 2004

To determine the optimum duration of follow-up for the assessment of drug efficacy against
Plasmodium falciparum
malaria, 96 trial arms from randomized controlled trials (RCTs) with follow-up of 28 days or longer that were conducted between 1990 and 2003 were analyzed. These trials enrolled 13,772 patients, and participating patients comprised 23% of all patients enrolled in RCTs over the past 40 years; 61 (64%) trial arms were conducted in areas where the rate of malaria transmission was low, and 58 (50%) trial arms were supported by parasite genotyping to distinguish true recrudescences from reinfections. The median overall failure rate reported was 10% (range, 0 to 47%). The widely used day 14 assessment had a sensitivity of between 0 and 37% in identifying treatment failures and had no predictive value. Assessment at day 28 had a sensitivity of 66% overall (28 to 100% in individual trials) but could be used to predict the true failure rate if either parasite genotyping was performed (
r
2
= 0.94) or if the entomological inoculation rate was known. In the assessment of drug efficacy against falciparum malaria, 28 days should be the minimum period of follow-up.
Heterologous Expression of the Enterococcal
vanA
Operon in Methicillin-Resistant
Staphylococcus aureus
Authors: Périchon Bruno , Courvalin Patrice
Published: October 2004

Two methicillin- and vancomycin-resistant
Staphylococcus aureus
strains, MI-VRSA and PA-VRSA, and
Enterococcus faecalis
DMC83006B, considered to be the potential donor of glycopeptide resistance to MI-VRSA, were studied. MI-VRSA is highly resistant to both glycopeptides, whereas PA-VRSA displays low-level resistance to vancomycin and reduced susceptibility to teicoplanin. We have analyzed the expression of the
vanA
operon in the three clinical isolates. Determination of the relative amounts of late peptidoglycan precursors and quantification of the
d,d
-peptidase activities, in the absence or after induction by glycopeptides, revealed that the resistance genes were expressed at similarly high levels in the three strains. Glycopeptide resistance stability in the three strains was studied by replica plating. Resistance was lost at high frequency, ca. 50%, after overnight growth of PA-VRSA in the absence of antibiotics, whereas it was fully stable in MI-VRSA and
E. faecalis
DMC83006B. Induction of resistance by vancomycin was significantly delayed in PA-VRSA relative to MI-VRSA. Low-level glycopeptide resistance of
S. aureus
PA-VRSA is thus likely due to instability of the genetic element, plasmid or transposon, carrying the
vanA
operon associated with a longer lag phase before growth resumes after induction by vancomycin.
Activity of the New Triazole Derivative Albaconazole against
Trypanosoma
(
Schizotrypanum
)
cruzi
in Dog Hosts
Authors: Guedes Paulo Marcos da Matta , Urbina Julio A. , de Lana Marta , Afonso Luis C. C. , Veloso Vanja M. , Tafuri Washington L. , Machado-Coelho George L. L. , Chiari Egler , Bahia Maria Terezinha
Published: October 2004

Albaconazole is an experimental triazole derivative with potent and broad-spectrum antifungal activity and a remarkably long half-life in dogs, monkeys, and humans. In the present work, we investigated the in vivo activity of this compound against two strains of the protozoan parasite
Trypanosoma
(
Schizotrypanum
)
cruzi
, the causative agent of Chagas' disease, using dogs as hosts. The
T. cruzi
strains used in the study were previously characterized (murine model) as susceptible (strain Berenice-78) and partially resistant (strain Y) to the drugs currently in clinical use, nifurtimox and benznidazole. Our results demonstrated that albaconazole is very effective in suppressing the proliferation of the parasite and preventing the death of infected animals. Furthermore, the parasitological, PCR, serological, and proliferative assay results indicated parasitological cure indices of 25 and 100% among animals inoculated with
T. cruzi
strain Y when they were treated with albaconazole at 1.5 mg/kg of body weight/day for 60 and 90 days, respectively. On the other hand, although albaconazole given at 1.5 mg/kg/day was very effective in suppressing the proliferation of the parasite in animals infected with the Berenice-78
T. cruzi
strain, no parasitological cure was observed among them, even when a longer treatment period (150 doses) was used. In conclusion, our results demonstrate that albaconazole has trypanocidal activity in vivo and is capable of inducing radical parasitological cure, although natural resistance to this compound was also indicated. Furthermore, the compound can be used in long-term treatment schemes (60 to 150 days) with minimal toxicity and thus represents a potentially useful candidate for the treatment of human Chagas' disease.
Rapid Direct Detection of Multiple Rifampin and Isoniazid Resistance Mutations in
Mycobacterium tuberculosis
in Respiratory Samples by Real-Time PCR
Authors: Marín Mercedes , de Viedma Darío García , Ruíz-Serrano María Jesús , Bouza Emilio
Published: October 2004

Rapid detection of resistance in
Mycobacterium tuberculosis
can optimize the efficacy of antituberculous therapy and control the transmission of resistant
M. tuberculosis
strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of
M. tuberculosis
strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant
M. tuberculosis
strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 10
3
CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-
M. tuberculosis
bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.
Mutations of
Pneumocystis jirovecii
Dihydrofolate Reductase Associated with Failure of Prophylaxis
Authors: Nahimana Aimable , Rabodonirina Meja , Bille Jacques , Francioli Patrick , Hauser Philippe M.
Published: October 2004

Most drugs used for prevention and treatment of
Pneumocystis jirovecii
pneumonia target enzymes involved in the biosynthesis of folic acid, i.e., dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). Emergence of
P. jirovecii
drug resistance has been suggested by the association between failure of prophylaxis with sulfa drugs and mutations in DHPS. However, data on the occurrence of mutations in DHFR, the target of trimethoprim and pyrimethamine, are scarce. We examined polymorphisms in
P. jirovecii
DHFR from 33 patients diagnosed with
P. jirovecii
pneumonia who were receiving prophylaxis with a DHFR inhibitor (
n
= 15), prophylaxis without a DHFR inhibitor (
n
= 11), or no prophylaxis (
n
= 7). Compared to the wild-type sequence present in GenBank, 19 DHFR nucleotide substitution sites were found in 18 patients with 3 synonymous and 16 nonsynonymous mutations. Of 16 amino acid changes, 6 were located in positions conserved among distant organisms, and five of these six positions are probably involved in the putative active sites of the enzyme. Patients with failure of prophylaxis, including a DHFR inhibitor, were more likely to harbor nonsynonymous DHFR mutations than those who did not receive such prophylaxis (9 of 15 patients versus 2 of 18;
P
= 0.008). Analysis of the rate of nonsynonymous versus synonymous mutations was consistent with selection of amino acid substitutions in patients with failure of prophylaxis including a DHFR inhibitor. The results suggest that
P. jirovecii
populations may evolve under selective pressure from DHFR inhibitors, in particular pyrimethamine, and that DHFR mutations may contribute to
P. jirovecii
drug resistance.
Potential for Interactions between Caspofungin and Nelfinavir or Rifampin
Authors: Stone Julie A. , Migoya Elizabeth M. , Hickey Lisa , Winchell Gregory A. , Deutsch Paul J. , Ghosh Kalyan , Freeman Amanda , Bi Sheng , Desai Rajesh , Dilzer Stacy C. , Lasseter Kenneth C. , Kraft Walter K. , Greenberg Howard , Waldman Scott A.
Published: October 2004

The potential for interactions between caspofungin and nelfinavir or rifampin was evaluated in two parallel-panel studies. In study A, healthy subjects received a 14-day course of caspofungin alone (50 mg administered intravenously [IV] once daily) (
n
= 10) or with nelfinavir (1,250 mg administered orally twice daily) (
n
= 9) or rifampin (600 mg administered orally once daily) (
n
= 10). In study B, 14 subjects received a 28-day course of rifampin (600 mg administered orally once daily), with caspofungin (50 mg administered IV once daily) coadministered on the last 14 days, and 12 subjects received a 14-day course of caspofungin alone (50 mg administered IV once daily). The coadministration/administration alone geometric mean ratio for the caspofungin area under the time-concentration profile calculated for the 24-h period following dosing [AUC
0-24
] was as follows (values in parentheses are 90% confidence intervals [CIs]): 1.08 (0.93-1.26) for nelfinavir, 1.12 (0.97-1.30) for rifampin (study A), and 1.01 (0.91-1.11) for rifampin (study B). The shape of the caspofungin plasma profile was altered by rifampin, resulting in a 14 to 31% reduction in the trough concentration at 24 h after dosing (
C
24h
), consistent with a net induction effect at steady state. Both the AUC and the
C
24h
were elevated in the initial days of rifampin coadministration in study A (61 and 170% elevations, respectively, on day 1) but not in study B, consistent with transient net inhibition prior to full induction. The coadministration/administration alone geometric mean ratio for the rifampin AUC
0-24
on day 14 was 1.07 (90% CI, 0.83-1.38). Nelfinavir does not meaningfully alter caspofungin pharmacokinetics. Rifampin both inhibits and induces caspofungin disposition, resulting in a reduced
C
24h
at steady state. An increase in the caspofungin dose to 70 mg, administered daily, should be considered when the drug is coadministered with rifampin.
Novel Approach to Characterization of Combined Pharmacodynamic Effects of Antimicrobial Agents
Authors: Tam Vincent H. , Schilling Amy N. , Lewis Russell E. , Melnick David A. , Boucher Adam N.
Published: October 2004

There is considerable need for new modeling approaches in the study of combined antimicrobial effects. Current methods based on the Loewe additivity and Bliss independence models are associated with implicit assumptions about the interacting system. To circumvent these limitations, we propose an alternative approach to the quantification of pharmacodynamic drug interaction (PDI). Pilot time-kill studies were performed with 10
8
CFU of
Pseudomonas aeruginosa
/ml at baseline with meropenem or tobramycin alone. The studies were repeated with 25 concentration combinations of meropenem (0 to 64 mg/liter) and tobramycin (0 to 32 mg/liter) in a five-by-five array. The data were modeled with a three-dimensional response surface using effect summation as the basis of null interaction. The interaction index (Ii) is defined as the ratio of the volumes under the planes (VUP) of the observed and expected surfaces: VUP
observed
/VUP
expected
. Synergy and antagonism are defined as Ii values of <1 and >1, respectively. In all combinations, an enhanced killing effect was seen compared to that of either drug at the same concentration. The most significant synergism was observed between 1 and 5 mg/liter of meropenem and between 1 and 4 mg/liter of tobramycin; seven out of nine combinations had a >2-log drop compared to the more potent agent. The Ii was found to be 0.76 (95% confidence interval, 0.65 to 0.91) for the concentration ranges of the agents. The results corroborate previous data indicating that meropenem is synergistic with an aminoglycoside when used in combination against
P. aeruginosa
. Our parametric approach to quantifying PDI appears robust and warrants further investigations.