In Vitro and In Vivo Inhibition of Murine Gamma Herpesvirus 68 Replication by Selected Antiviral Agents

We have evaluated the susceptibility of the murine gamma herpesvirus 68 (MHV-68) to a variety of antiviral agents. The acyclic nucleoside phosphonate analogs cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and adefovir [9-(2-phosphonylmethoxyethyl)adenine] efficiently inhibited the replication of the virus in Vero cells (50% effective concentrations [EC₅₀s], 0.008, 0.06, and 2.2 μg/ml, respectively). Acyclovir, ganciclovir, and brivudin [(E)-5-(2-bromovinyl)-2′-deoxyuridine] had equipotent activities (EC₅₀s, 1.5 to 8 μg/ml), whereas foscarnet and penciclovir were less effective (EC₅₀s, 23 and ≥30 μg/ml, respectively). The novel N-7-substituted nucleoside analog S2242 [7-(1,3-dihydroxy-2-propoxymethyl)purine] inhibited MHV-68 replication by 50% at 0.2 μg/ml. The susceptibilities of MHV-68 and Epstein-Barr virus (EBV) to cidofovir, HPMPA, adefovir, and acyclovir were found to be comparable. However, for penciclovir, ganciclovir, brivudin, and S2242, major differences in the sensitivity of MHV-68 and EBV were observed, suggesting that MHV-68 is not always an optimal surrogate for the study of antiviral strategies for EBV. When evaluated with a model for lethal MHV-68 infections in mice with severe combined immunodeficiency, cidofovir proved to be very efficient in protecting against virus-induced mortality (100% survival at 50 days postinfection), whereas acyclovir, brivudin, and adefovir had little or no effect.     

Rapid Ganciclovir Susceptibility Assay Using Flow Cytometry for Human Cytomegalovirus Clinical Isolates

Rapid, quantitative, and objective determination of the susceptibilities of human cytomegalovirus (HCMV) clinical isolates to ganciclovir has been assessed by an assay that uses a fluorochrome-labeled monoclonal antibody to an HCMV immediate-early antigen and flow cytometry. Analysis of the ganciclovir susceptibilities of 25 phenotypically characterized clinical isolates by flow cytometry demonstrated that the 50% inhibitory concentrations (IC₅₀s) of ganciclovir for 19 of the isolates were between 1.14 and 6.66 μM, with a mean of 4.32 μM (±1.93) (sensitive; IC₅₀ less than 7 μM), the IC₅₀s for 2 isolates were 8.48 and 9.79 μM (partially resistant), and the IC₅₀s for 4 isolates were greater than 96 μM (resistant). Comparative analysis of the drug susceptibilities of these clinical isolates by the plaque reduction assay gave IC₅₀s of less than 6 μM, with a mean of 2.88 μM (±1.40) for the 19 drug-sensitive isolates, IC₅₀s of 6 to 8 μM for the partially resistant isolates, and IC₅₀s of greater than 12 μM for the four resistant clinical isolates. Comparison of the IC₅₀s for the drug-susceptible and partially resistant clinical isolates obtained by the flow cytometry assay with the IC₅₀s obtained by the plaque reduction assay showed an acceptable correlation (r² = 0.473; P = 0.001), suggesting that the flow cytometry assay could substitute for the more labor-intensive, subjective, and time-consuming plaque reduction assay.     

Erythromycin Resistance Genes in Group A Streptococci in Finland

Streptococcus pyogenes isolates (group A streptococcus) of different erythromycin resistance phenotypes were collected from all over Finland in 1994 and 1995 and studied; they were evaluated for their susceptibilities to 14 antimicrobial agents (396 isolates) and the presence of different erythromycin resistance genes (45 isolates). The erythromycin-resistant isolates with the macrolide-resistant but lincosamide- and streptogramin B-susceptible phenotype (M phenotype) were further studied for their plasmid contents and the transferability of resistance genes. Resistance to antimicrobial agents other than macrolides, clindamycin, tetracycline, and chloramphenicol was not found. When compared to our previous study performed in 1990, the rate of resistance to tetracycline increased from 10 to 93% among isolates with the inducible resistance (IR) phenotype of macrolide, lincosamide, and streptogramin B (MLS_B) resistance. Tetracycline resistance was also found among 75% of the MLS_B-resistant isolates with the constitutive resistance (CR) phenotype. Resistance to chloramphenicol was found for the first time in S. pyogenes in Finland; 3% of the isolates with the IR phenotype were resistant. All the chloramphenicol-resistant isolates were also resistant to tetracycline. Detection of erythromycin resistance genes by PCR indicated that, with the exception of one isolate with the CR phenotype, all M-phenotype isolates had the macrolide efflux (mefA) gene and all the MLS_B-resistant isolates had the erythromycin resistance methylase (ermTR) gene; the isolate with the CR phenotype contained the ermB gene. No plasmid DNA could be isolated from the M-phenotype isolates, but the mefA gene was transferred by conjugation.     

Update on treatment of CMV retinitis

Recent developments in the treatment of CMV retinitis are highlighted, including new methods of administering ganciclovir, and use of a new parenteral agent, cidofovir (also called HPMPC). An intraocular ganciclovir sustained-release device has been developed and tested in two different trials. This implant releases 6 mg of ganciclovir over 33 to 39 weeks. In a study randomizing eyes of thirty patients with CMV retinitis, time to disease progression was 226 days for the immediate treatment group compared to 15 days in the deferred group; adverse effects are summarized. In a second study, comparing intraocular to intravenous ganciclovir in CMV retinitis patients, implant patients had a significantly longer time to retinitis progression. Significant risks to vision were noted, however, in the implant group. In another immediate vs. deferred retinitis trial, immediate intravenous cidofovir was shown to be effective, with a six-fold increase in time to progression over those who first received therapy after evidence of first progression. The primary adverse effect of intravenous cidofovir was nephrotoxicity, which was ameliorated by hydration and probenecid. Intravitreal cidofovir was studied, also resulting in delayed progression of retinitis. Finally, the use of oral ganciclovir to prevent CMV end-organ disease resulted in a decrease of CMV infections by about fifty percent, but much controversy remains due to the involvement of multiple individual factors.     

Characterization of Vancomycin-Resistant Enterococcus faecium Isolates from the United States and Their Susceptibility In Vitro to Dalfopristin-Quinupristin

In the course of clinical studies with the investigational streptogramin antimicrobial dalfopristin-quinupristin, isolates of vancomycin-resistant Enterococcus faecium were referred to our laboratory from across the United States. Seventy-two percent of the strains were of the VanA type, phenotypically and genotypically, while 28% were of the VanB type. High-level resistance to streptomycin or gentamicin was observed in 86 and 81%, respectively, of the VanA strains but in only 69 and 66%, respectively, of the VanB strains. These enterococci were resistant to ampicillin (MIC for 50% of the isolates tested [MIC₅₀] and MIC₉₀, 128 and 256 μg/ml, respectively) and to the other approved agents tested, with the exception of chloramphenicol (MIC₉₀, 8 μg/ml) and novobiocin (MIC₉₀, 1 μg/ml). Considering all of the isolates submitted, dalfopristin-quinupristin inhibited 86.4% of them at concentrations of ≤1 μg/ml and 95.1% of them at ≤2 μg/ml. However, for the data set comprised of only the first isolate submitted for each patient, 94.3% of the strains were inhibited at concentrations of ≤1 μg/ml and 98.9% were inhibited at concentrations of ≤2 μg/ml. Multiple drug resistance was very common among these isolates of vancomycin-resistant E. faecium, while dalfopristin-quinupristin inhibited the majority at concentrations that are likely to be clinically relevant.     

Pharmacokinetics and Safety of a New Parenteral Carbapenem Antibiotic, Biapenem (L-627), in Elderly Subjects

The pharmacokinetics and tolerability of a new parenteral carbapenem antibiotic, biapenem (L-627), were studied in healthy elderly volunteers aged 65 to 74 years (71.6 ± 2.7 years [mean ± standard deviation], n = 5; group B) and ≥75 years (77.8 ± 1.9 years, n = 5; group C), following single intravenous doses (300 and 600 mg), and compared with those of healthy young male volunteers aged 20 to 29 years (23.0 ± 3.5 years, n = 5; group A). The agent was well tolerated in all three age groups. Serial blood and urine samples were analyzed for biapenem to obtain key pharmacokinetic parameters by both two-compartment model-dependent and -independent methods. The maximum plasma concentration and area under plasma concentration-versus-time curve (AUC) increased in proportion to the dose in all three groups. Statistically significant age-related effects for AUC, total body clearance, and renal clearance (CL_R) were found, while elimination half-life (t_1/2β) and percent cumulative recovery from urine of unchanged drug (% UR) remained unaltered (t_1/2β, 1.51 ± 0.42 [300 mg] and 2.19 ± 0.64 [600 mg] h [group A], 1.82 ± 1.14 and 1.45 ± 0.36 h [group B], and 1.75 ± 0.23 and 1.59 ± 0.18 h [group C]; %UR, 52.6% ± 3.0% [300 mg] and 53.1% ± 5.1% [600 mg] [group A], 46.7% ± 7.4% and 53.0% ± 4.8% [group B], and 50.1% ± 5.2% and 47.1% ± 7.6% [group C]). A significant linear correlation was observed between the CL_R of biapenem and creatinine clearance at the dose of 300 mg but not at 600 mg. The steady-state volume of distribution tended to be decreased with age, although not significantly. Therefore, the age-related changes in parameters of biapenem described above were attributable to the combination of decreased lean body mass and lowered renal function of the elderly subjects. However, the magnitude of those changes does not necessitate dosage adjustment in elderly patients with normal renal function for their age.     

Clinical Pharmacokinetics of 1-[((S)-2-Hydroxy-2-Oxo-1,4,2-Dioxaphosphorinan-5-yl)methyl]cytosine in Human Immunodeficiency Virus-Infected Patients

The pharmacokinetics and bioavailability of 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine (cyclic HPMPC) were examined at four doses in 22 patients with human immunodeficiency virus infection. Two groups of six patients received a single dose of cyclic HPMPC at 1.5 or 3.0 mg/kg of body weight by each of the oral and intravenous routes in a random order with a 2-week washout period between administrations. Additional patients received single intravenous doses of cyclic HPMPC at 5.0 mg/kg (n = 6) or 7.5 mg/kg (n = 4). Serial serum and urine samples were collected at intervals over 24 h after dosing. The concentrations of cyclic HPMPC and cidofovir in serum and urine samples were determined by validated reverse-phase ion-pairing high-performance liquid chromatography methods with derivatization and fluorescence detection. After intravenous administration of cyclic HPMPC, concentrations of cyclic HPMPC declined in a biexponential manner, with a mean ± standard deviation half-life of 1.09 ± 0.12 h (n = 22). The pharmacokinetics of cyclic HPMPC were independent of dose over the dose range of 1.5 to 7.5 mg/kg. The total clearance of cyclic HPMPC from serum and the volume of distribution of intravenous cyclic HPMPC were 198 ± 39.6 ml/h/kg and 338 ± 65.1 ml/kg, respectively (n = 22). The renal clearance of cyclic HPMPC (132 ± 27.3 ml/h/kg; n = 22) exceeded the creatinine clearance (86.2 ± 16.3 ml/h/kg), indicating active tubular secretion. The cyclic HPMPC excreted in urine in 24 h accounted for 71.3% ± 16.0% of the administered dose. Cidofovir was formed from cyclic HPMPC in vivo with a time to the maximum concentration in serum of 1.64 ± 0.23 h (n = 22). Cidofovir levels declined in an apparent monoexponential manner, with a mean terminal half-life of 3.98 ± 1.26 h (n = 22). The cidofovir excreted in urine in 24 h accounted for 9.40% ± 2.33% of the administered cyclic HPMPC dose. Exposure to cidofovir after intravenous administration of cyclic HPMPC was dose proportional and was 14.9% of that from an equivalent dose of cidofovir. The present study suggests that intravenous cyclic HPMPC also has a lower potential for nephrotoxicity in humans compared to that of intravenous cidofovir. The oral bioavailabilities of cyclic HPMPC were 1.76% ± 1.48% and 3.10% ± 1.16% with the administration of doses of 1.5 and 3.0 mg/kg, respectively (n = 6 per dose). The maximum concentrations of cyclic HPMPC in serum were 0.036 ± 0.021 and 0.082 ± 0.038 μg/ml after the oral administration of doses of 1.5 and 3.0 mg/kg, respectively. Cidofovir reached quantifiable levels in the serum of only one patient for each of the 1.5- and 3.0-mg/kg oral cyclic HPMPC doses.     

Foscarnet treatment of cytomegalovirus retinitis in patients with the acquired immunodeficiency syndrome.

Ten patients with acquired immunodeficiency syndrome with newly diagnosed cytomegalovirus (CMV) retinitis were treated with an induction regimen of intravenous foscarnet, 60 mg/kg of body weight, administered as a 2-h infusion and repeated every 8 h for 14 days. At the end of induction, 9 of 10 patients had stabilized (no new retinal lesions and stable old lesions [7 patients]) or improved (decreased retinal opacification [2 patients]). All eight patients with CMV in urine or blood upon entry into the study had negative urine and blood cultures at the end of induction. After induction therapy, seven patients continued maintenance foscarnet therapy, 60 mg/kg as a single daily infusion, 5 days/week. In six patients, retinal lesions increased in size after 2 to 32 weeks of maintenance therapy. One was invaluable because a retinal detachment developed. Only 9 of 42 blood and urine cultures obtained during maintenance foscarnet therapy yielded CMV, compared with 7 of 14 obtained prior to the initiation of foscarnet induction therapy (P = 0.04). Foscarnet toxicity was mild and infrequent: elevation in serum creatinine by 0.5 to 1.3 mg/dl over the base line (two patients), muscle twitching (three patients), hemoglobin decrease by 1 mg/dl (two patients), nausea (two patients), absolute neutrophil count decrease by 50% (one patient), rise in serum phosphorus to greater than 5.5 mg/dl (four patients), and proteinuria (two patients). Intermittently administered intravenous foscarnet appears to be an effective, relatively nontoxic therapy for CMV retinitis. Additional studies to determine the optimal dosage for maintenance therapy are needed, as are comparative trials with ganciclovir.     

Pharmacokinetics and Absolute Bioavailability of Oral Foscarnet in Human Immunodeficiency Virus-Seropositive Patients

The pharmacokinetics, absolute bioavailability, accumulation, and tolerability over 8 days of an oral formulation of foscarnet (90 mg/kg of body weight once daily [QD] [n = 6], 90 mg/kg twice daily [BID] [n = 6], and 180 mg/kg QD [n = 3]) were investigated in 15 asymptomatic, human immunodeficiency virus-seropositive male patients free of active cytomegalovirus infection and with normal upper gastrointestinal function. Peak plasma drug concentrations were (mean ± standard deviation) 46.4 ± 10.8 μM (90 mg/kg QD), 45.7 ± 6.9 μM (90 mg/kg BID), and 64.9 ± 31.7 μM (180 mg/kg QD) on day 1 and rose to 86.2 ± 35.8, 78.7 ± 35.2, and 86.4 ± 25.0 μM, respectively, on day 8. The mean peak concentration in plasma following the intravenous administration of foscarnet (90 mg/kg) was 887.3 ± 102.7 μM (n = 13). The terminal half-life in plasma remained unchanged, averaging 5.5 ± 2.2 h on day 1 (n = 15) and 6.6 ± 1.9 h on day 8 (n = 13), whereas it was 5.7 ± 0.7 h following intravenous dosing. Oral bioavailabilities were 9.1% ± 2.2% (90 mg/kg QD), 9.5% ± 1.7% (90 mg/kg BID), and 7.6% ± 3.7% (180 mg/kg QD); the accumulation ratios on the 8th day of dosing were 2.1 ± 1.1, 1.8 ± 0.4, and 1.7 ± 0.7, respectively. The overall 24-h urinary excretion of oral foscarnet averaged 7.8% ± 2.6% (day 1) and 13.4% ± 6.0% (day 8), whereas it was 95.0% ± 4.9% after intravenous dosing. The glomerular filtration rate and creatinine clearance remained constant, and the mean 24-h renal clearances of foscarnet for the entire study group were 96 ± 18 ml/min (day 1), 88 ± 13 ml/min (day 8), and 103 ± 16 ml/min after intravenous dosing. Adverse effects were largely confined to gastrointestinal disturbances, with all subjects experiencing diarrhea that was dose dependent in its severity. The results suggest that the formulation studied would require significant improvement with respect to tolerability and bioavailability to gain clinical acceptance.     

Detection of grlA and gyrA Mutations in 344 Staphylococcus aureus Strains

Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC→TTC or TAC (Ser-80→Phe or Tyr) and the gyrA mutation of TCA→TTA (Ser-84→Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. The grlA point mutations of CAT→CAC (His-77 [silent]), TCA→CCA (Ser-81→Pro), and ATA→ATT (Ile-100 [silent]) were novel, as was the GAC→GGC (Asp-73→Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC ≥ 3.13 μg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC₅₀] = 50 μg/ml). Those with the Ser-80→Phe or Tyr alteration in grlA but wild-type gyrA showed a lower level of ciprofloxacin resistance (MIC₅₀ ≤ 12.5 μg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC ≤ 6.25 μg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was ≤6.25 μg/ml.     

Genotypic characterization of the DNA polymerase and sensitivity to antiviral compounds of foscarnet-resistant herpes simplex virus type 1 (HSV-1) derived from a foscarnet-sensitive HSV-1 strain

Foscarnet is widely used for the treatment of acyclovir-resistant herpesvirus infections, and foscarnet-resistant herpesvirus infections are a serious concern in immunocompromised patients. Twenty-seven single-plaque isolates of herpes simplex virus type 1 (HSV-1) resistant to foscarnet were selected from foscarnet- and acyclovir-sensitive HSV-1 strain TAS by exposure to foscarnet, and the DNA polymerase genes were analyzed. The sensitivities of these mutants to foscarnet, cidofovir, S2242, acyclovir, ganciclovir, and penciclovir were determined. A single amino acid substitution, double amino acid substitutions, and a combination of a single amino acid substitution with a deletion or insertion of amino acid residues in the viral DNA polymerase were demonstrated in 21, 4, and 2 isolates, respectively. Of the 27 isolates, an amino acid substitution of serine for asparagine at amino acid position 724 in the DNA polymerase (724 S-N) was detected in 8 isolates. An amino acid substitution in conserved region II was demonstrated in these eight isolates as well as four other isolates. The mutation in the DNA polymerase responsible for resistance to foscarnet was located between the pre-IV region and conserved region V, especially within conserved region II. All the isolates were sensitive or hypersensitive to cidofovir and ganciclovir. Seven, 5, and 15 of the 27 isolates were also sensitive to S2242, acyclovir, and penciclovir, respectively. Thus, most of the foscarnet-resistant HSV-1 isolates were sensitive or hypersensitive to cidofovir and ganciclovir.     

Ganciclovir sensitivity of cytomegalovirus at diagnosis and during treatment of cytomegalovirus pneumonia in marrow transplant recipients.

Cytomegalovirus (CMV) often persists in the lungs of marrow transplant patients with CMV pneumonia, despite ganciclovir (GCV) treatment. To determine whether GCV resistance contributes to viral persistence, the susceptibilities of CMV isolates from diagnostic bronchoalveolar lavage samples and CMV isolates obtained during treatment or from autopsy lung tissue from 12 patients were compared by DNA hybridization. Resistance (50% effective dose, > 12 microM) was detected in an isolate from only one patient who had also received several courses of GCV. GCV resistance did not explain the persistence of CMV in the lung.     

Human Cytomegalovirus Resistance to Deoxyribosylindole Nucleosides Maps to a Transversion Mutation in the Terminase Subunit-Encoding Gene UL89

Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC₅₀] = 0.34 μM) than ganciclovir (EC₅₀ = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC₅₀ = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC₅₀ = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC₅₀ for wild-type HCMV = 0.25 ± 0.04 μM, EC₅₀ for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).     

Iritis associated with intravenous cidofovir

OBJECTIVE: To report two patients with AIDS and cytomegalovirus retinitis who developed iritis after receiving intravenous cidofovir. Both experienced recurrent symptoms upon rechallenge. CASE SUMMARIES: Two HIV-positive patients with cytomegalovirus retinitis infections previously controlled with intravenous ganciclovir or foscarnet were treated with intravenous cidofovir. Symptoms of iritis developed after the second or third dose of cidofovir. One patient experienced symptoms unilaterally, while the other patient had bilateral symptoms. In both patients, the iritis resolved with topical ophthalmic therapy, but recurred following subsequent infusions of cidofovir. Therapy with cidofovir was discontinued, and no further recurrences of iritis were noted. One patient had post-inflammatory fixed dilated pupils. CONCLUSIONS: Iritis can uncommonly occur in patients receiving intravenous cidofovir and oral probenecid. With prompt drug discontinuation and administration of topical corticosteroids and/or mydriatic agents, symptoms are usually reversible.     

Recombinant Phenotyping of Cytomegalovirus UL97 Kinase Sequence Variants for Ganciclovir Resistance

A strain of human cytomegalovirus, T2211, modified from standard laboratory strain AD169 to contain a secreted alkaline phosphatase reporter gene for rapid viral quantitation, was cloned as a bacterial artificial chromosome, BA1, and then mutagenized to create recombinant viruses containing viral UL97 kinase sequence variants found in clinical specimens after ganciclovir treatment, but with no phenotypic data to determine their role in drug resistance. Seven control strains and 14 other recombinant strains were phenotyped for ganciclovir resistance and compared with similar strains created using prior technology to show a good concordance of findings. Sequence changes V466M, H469Y, A478V, N510S, A588V, K599R, L600I, G623S, T659I, and V665I were found to confer no significant ganciclovir resistance, while mutations L405P, M460T, A594E, and C603R conferred 3- to 9-fold increases in ganciclovir 50% inhibitory concentrations. Different mutations at codons 594 (A594V, A594E) and 603 (C603W, C603S) conferred varied amounts of ganciclovir resistance. Advances in recombinant phenotyping make it easier to show that many uncharacterized UL97 sequence variants do not confer ganciclovir resistance, but some are newly confirmed as resistance associated, including one (L405P) which is outside the codon range where such mutations are usually found. This information should improve the interpretation of genotypic data generated by diagnostic laboratories.Prolonged use of ganciclovir (GCV) or its prodrug valganciclovir, often for several months in transplant recipients undergoing primary cytomegalovirus (CMV) infection, may result in the development of drug resistance, which is suspected when circulating viral loads increase during ongoing therapy (17). In current clinical practice, GCV resistance is usually confirmed by analysis of CMV DNA sequences amplified from posttreatment clinical specimens, since viral culture isolates or pretreatment baseline CMV sequences are not commonly available. GCV resistance typically results from mutations in the viral UL97 kinase gene responsible for the initial phosphorylation of GCV, but sometimes mutations in the viral UL54 DNA polymerase (pol) gene emerge to confer added GCV resistance and cross-resistance to cidofovir and/or foscarnet. With both genes, an extensive and evolving database of mutations related to drug resistance has been documented and used for interpretation of genotypic test results.In the UL97 gene, a number of well-defined (“canonical”) mutations at codons 460, 520, and 590 to 607 are found in GCV-resistant clinical strains, with mutations M460V/I, H520Q, C592G, A594V, L595S, and C603W occurring most frequently (6). Point and deletion mutations in the codon range 590 to 607 confer various degrees of GCV resistance, while other changes in this region confer no significant resistance (6). Noncanonical UL97 sequence changes have repeatedly been found in patients who have received GCV. Sequences of pretreatment and/or drug-susceptible CMV isolates are available to indicate that certain sequence variants are probably normal interstrain polymorphisms unrelated to drug resistance (26), but in many cases, insufficient data exist to determine if the sequence variants represent polymorphisms, resistance-related mutations, or reporting errors (6, 16, 26, 32). Speculation about the significance of uncharacterized UL97 sequence variants has resulted in some misleading attributions of GCV resistance (5, 31), which can be confusing to clinicians and diagnostic laboratories.The role of viral gene mutations in drug resistance is determined by recombinant phenotyping (marker transfer), whereby a specific sequence change is transferred to a reference laboratory strain of CMV and the resulting effect on drug susceptibility is assessed by the drug concentration required to reduce viral growth by 50% (EC₅₀). Confirmed UL97 GCV resistance mutations usually confer 3- to 15-fold increases in the GCV EC₅₀ (6). Many UL97 sequence changes have not been phenotyped, mainly because the size of the CMV genome makes its site-specific mutagenesis technically nontrivial. In recent years, however, the work has benefited from use of standard laboratory CMV strains modified with reporter genes to facilitate viral quantitation (10) and mutagenesis of the CMV genome as a bacterial artificial chromosome (BAC) (4, 27). Here, we combine these advances to determine the significance of many UL97 sequence changes described in the existing literature or encountered more recently in diagnostic laboratories.     

Axenically Grown Amastigotes of Leishmania infantum Used as an In Vitro Model To Investigate the Pentavalent Antimony Mode of Action

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC₅₀s], about 1.8 mg/ml and 22 μg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC₅₀s, of 0.54 and 1.32 μg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.     

Widespread dissemination of aminoglycoside resistance genes armA and rmtB in Klebsiella pneumoniae isolates in Taiwan producing CTX-M-type extended-spectrum beta-lactamases

Among 235 extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL) isolates collected from a nationwide surveillance performed in Taiwan, 102 (43.4%) were resistant to amikacin. Ninety-two of these 102 (90.2%) isolates were carrying CTX-M-type β-lactamases individually or concomitantly with SHV-type or CMY-2 β-lactamases. The armA and rmtB alleles were individually detected in 44 and 37 of these 92 isolates, respectively. One isolate contained both armA and rmtB. The coexistence of the aac(6′)-Il and rmtB genes was detected in three isolates. CTX-M-type β-lactamase genes belonging to either group 1 (CTX-M-3 and CTX-M-15) or group 9 (CTX-M-14) were found in all armA- or rmtB-bearing ESBL-producing K. pneumoniae isolates, and all were conjugatively transferable. All except one of the isolates bearing armA produced CTX-M enzymes of group 1, and the remaining isolate bearing armA produced a group 9 CTX-M-type β-lactamase. On the contrary, in the majority of rmtB carriers, the CTX-M-type β-lactamase belonged to group 9 (62.2%). Molecular typing revealed that the amikacin-resistant ESBL-producing K. pneumoniae isolates were epidemiologically unrelated, indicating that the acquisition of resistance was not through the spread of a resistant clone or a resistance plasmid. A tandem repeat or multiple copies of bla_CTX-M-3 were found in some armA-bearing isolates. An ISEcp1 insert was found in all CTX-M ESBL-producing K. pneumoniae isolates carrying armA or rmtB. In conclusion, the concomitant presence of a 16S rRNA methylase gene (armA or rmtB) and bla_CTX-M among amikacin-resistant ESBL-producing K. pneumoniae isolates is widespread in Taiwan.     

Failure of adjunctive cytomegalovirus intravenous immune globulin to improve efficacy of ganciclovir in patients with acquired immunodeficiency syndrome and cytomegalovirus retinitis: a phase 1 study.

Six men with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus (CMV) retinitis, treated with combined ganciclovir induction therapy and hyperimmune globulin (CMV-IGIV) for 10 days followed by CMV-IGIV alone, had a median time to retinitis progression shorter (7 days) than had eight historical controls given ganciclovir maintenance therapy (54 days; P = 0.06) and similar to that in eight controls given ganciclovir for 10 days only (19 days; P = 0.97). CMV-IGIV, which also failed to inhibit CMV replication in blood and urine, did not appear to add markedly to the efficacy of ganciclovir in acquired immunodeficiency syndrome-associated CMV retinitis.     

In Vitro Susceptibilities of Bordetella pertussis and Bordetella parapertussis to Two Ketolides (HMR 3004 and HMR 3647), Four Macrolides (Azithromycin, Clarithromycin, Erythromycin A, and Roxithromycin), and Two Ansamycins (Rifampin and Rifapentine)

When tested by agar dilution on Mueller-Hinton agar supplemented with 5% horse blood, the ketolides HMR 3004 and HMR 3647 were slightly more active (MIC at which 90% of the isolates were inhibited [MIC₉₀], 0.03 μg/ml) against Bordetella pertussis than azithromycin, clarithromycin, erythromycin A, and roxithromycin. Azithromycin (MIC₉₀, 0.06 μg/ml) was the most active compound against B. parapertussis. Rifampin and rifapentine were considerably less active.     

Identification and Expression of Multidrug Transporters Responsible for Fluconazole Resistance in Candida dubliniensis

Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [³H]fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible Δpdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [³H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.