Detection and pharmacokinetics of a cytomegalovirus (CMV) DNA plasmid in human plasma during a clinical trial of an intramuscular CMV vaccine in hematopoietic stem cell transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) causes significant morbidity and mortality in solid organ and bone marrow transplant recipients. DNA vaccines can provide both humoral and cellular immunity without exposing immune-compromised persons to replication-competent CMV. METHODS: We studied the kinetics of CMV vaccine DNA in plasma. The samples were obtained from vaccine recipients who were enrolled in a double-blinded, placebo-controlled clinical trial of an intramuscular, plasmid-based, bivalent DNA vaccine for CMV in stem cell transplant recipients. Residual specimens on patients enrolled in the vaccine trial were saved until the trial was unblinded and published. Quantitative real-time polymerase chain reaction (PCR) was used to detect and quantify CMV glycoprotein B (gB) DNA in plasma from 4 recipients of the vaccine. The melting temperature of the vaccine gB amplicon was 62.4°C, compared to 68.8°C, which is seen with the wild-type virus. RESULTS: Sequence analysis revealed that there were 3 mismatches between the fluorescent resonance energy transfer probe and the vaccine DNA sequence. CONCLUSION: Because preemptive treatment of CMV disease in stem cell transplant patients is based on quantitative PCR analysis of viral sequences in plasma, it is important that vaccine sequences not be confused with those in wild-type virus. Confusion could lead to treatment with toxic medications, potentially compromising the transplant. Effects of PCR target choice and amplicon detection techniques on patient management and vaccine trials are discussed.

Vaccination adherence in hematopoietic stem cell transplant patients: a pilot study on the impact of vaccination cards and reminder telephone calls.

BACKGROUND: The Karmanos Cancer Center Bone Marrow Transplant (KCC BMT) Center updated the vaccination protocol for transplant patients based on joint guidelines recently published by European Bone Marrow Transplantation, Centers for Disease Control, Infectious Diseases Society of America, and the American Society for Bone Marrow Transplantation. The objectives for this study were to determine the impact of vaccination cards and telephone outreach on vaccine adherence and to determine the reasons for missed or delayed vaccinations. METHODS: Retrospective analysis consisted of autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients at the KCC BMT Clinic who received vaccines based on the guidelines published in 2009. Adherence was calculated as percentage of vaccines missed over the vaccination period. RESULTS: Overall, 37 of 111 patients (33%) missed at least 1 vaccine set and 29 patients (26%) received 1 set later than recommended. Reasons for missed and delayed vaccines included neutropenia/thrombocytopenia, anticoagulation, active infection, and graft-versus-host complications. CONCLUSIONS: Current guidelines recommend a complex vaccination schedule for the HSCT recipients, which may lead to difficulties with adherence. Our study shows that missed and delayed vaccinations are common in both autologous and allogeneic HSCT recipients. Methods to improve adherence are needed.

In vitro-in vivo correlation of voriconazole resistance due to G448S mutation (cyp51A gene) in Aspergillus fumigatus.

Invasive pulmonary aspergillosis continues to be associated with a high mortality despite timely and appropriate therapy. Although host immunity plays a major role in poor clinical response, antifungal drug resistance cannot be ignored. Our studies were aimed 1) to study the mechanism of drug resistance in voriconazole strains of Aspergillus fumigatus, 2) to establish a causal relationship between cyp51A mutation and voriconazole resistance (VRC-R), and 3) to determine whether VRC-R due to cyp51A mutation correlated with in vivo resistance. A point mutation (G448S) involving cyp51A gene in VRC-R isolate was associated with resistance to VRC but not to posaconazole (POS); POS had superior activity to VRC in reducing lung fungal burden and mortality in mice infected with a VRC-R mutant of A. fumigatus. Our study demonstrated that azole resistance is based on specific site of cyp51A mutation and that in vitro VRC-R correlates with in vivo resistance.

Clinical impact of the use of 16S rRNA sequencing method for the identification of "difficult-to-identify" bacteria in immunocompromised hosts.

Molecular method of 16S rRNA sequencing is reported to be helpful in the accurate identification of organisms with ambiguous phenotypic profiles. We analyzed the use of 16S rRNA sequencing method to identify clinically significant, "difficult-to-identify" bacteria recovered from clinical specimens, and evaluated its role in patient management and consequent clinical outcome. Among the 172 "difficult-to-identify" bacteria recovered over a 4-year period, 140 were gram-positive cocci or gram-negative bacilli; identification by 16S rRNA did not play a role in the management of patients infected with these bacteria. From 32 patients, 33 "difficult-to-identify" gram-positive bacilli were identified; the organisms were mycobacteria, Nocardia, Tsukamurella, Rhodococcus, and Gordonia. In 24 patients for whom clinical data were available, results from the 16S rRNA sequencing method led to treatment change in 14 immunocompromised patients (including 7 hematopoietic stem cell recipients and 1 liver transplant recipient). Therapy was modified in 9 patients, initiated in 3 patients, and discontinued in 2 patients. Most patients' therapy was switched to oral antibiotics with discontinuation of intravascular catheters, facilitating early hospital discharge. All 14 patients were alive 30 days after infection onset. The present study demonstrates the clinical application of 16S rRNA sequencing method to identify "difficult-to-identify" mycobacteria and other gram-positive bacilli in clinical specimens, particularly in immunocompromised hosts.

Weissella confusa: an unexpected cause of vancomycin-resistant gram-positive bacteremia in immunocompromised hosts.

We report the first case of Weissella confusa bacteremia in an allogeneic hematopoietic stem cell transplant patient. After engraftment and discharge, the patient returned with fever and graft failure and was started on an empiric regimen of aztreonam and vancomycin. A blood culture grew an alpha-hemolytic, gram-positive coccus forming pairs and chains, originally thought to be a viridans Streptococcus and a skin contaminant. The isolation of the organism from multiple blood cultures, and the presence of vancomycin resistance prompted identification and additional susceptibility testing. The RapID(™) Str panel, which has W. confusa in its database, provided multiple incorrect identifications. The MicroScan WalkAway 96 SI, using PC-20 or -29 panels, also did not identify this bacterium, because it is not in their database. The organism was identified as W. confusa by 16S rDNA sequencing. Antibiotic susceptibility determination by Etest revealed vancomycin resistance and daptomycin susceptibility. Therapy was changed to daptomycin, and the infection resolved. Additionally, W. confusa sepsis, with multiple positive blood cultures, developed in a patient in the burn unit at our medical center. The patient's blood cultures remained positive until vancomycin was discontinued and daptomycin therapy initiated. Infections with vancomycin-resistant, gram-positive cocci are emerging among immuno compromised hosts. Under appropriate circumstances, clinicians need to request that the laboratory perform susceptibility testing and accurate identification, by nucleic acid sequencing if necessary. Sequencing of 16S rDNA is an important tool in the accurate identification of unusual pathogens.

Listeria grayi: vancomycin-resistant, gram-positive rod causing bacteremia in a stem cell transplant recipient.

We report the first case of Listeria grayi bacteremia in a stem cell transplant recipient. The patient developed bacteremia with a gram-positive rod that was initially thought to be Corynebacterium species and a skin contaminant. The organism grew in multiple blood cultures and therapy with vancomycin was initiated. The API Coryne (version 3.0) identified the organism as L. grayi. Susceptibility testing by Etest suggested that the organism was resistant to vancomycin, but susceptible to ampicillin. After therapeutic change from vancomycin to ampicillin, the bacteremia cleared. Empiric therapy with vancomycin for all gram-positive bacterial infections is not appropriate. Accurate identification and antibiotic susceptibility is important, particularly in those with persistent bacteremia.

In vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes.

OBJECTIVES: Zygomycosis is an uncommon but devastating disease with few therapeutic options. Calcineurin inhibitors and sirolimus (mTOR inhibitor), commonly used in transplant patients as immunosuppressives, have antifungal activity. They are known to demonstrate synergy with triazoles against certain fungi, though limited data exist about their activity against zygomycetes. Our aim was to study the in vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes. METHODS: Drug interactions were assessed with chequerboard dilution for posaconazole with calcineurin inhibitors and sirolimus according to the CLSI M38-A2 method for filamentous fungi. Twenty-eight clinical isolates were studied, including Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, Mucor sp., Cunninghamella bertholletiae, Myocladus corymbifera and Apophysomyces elegans. Combinations of posaconazole with tacrolimus, cyclosporin A or sirolimus were used. Experiments were performed in duplicate. Mean fractional inhibitory concentration indices were calculated. RESULTS: Posaconazole with calcineurin inhibitors demonstrated consistent synergy against C. bertholletiae, M. corymbifera and A. elegans, whereas synergy or no interaction was primarily observed against R. arrhizus, R. microsporus, R. pusillus and Mucor. Antagonism was seen with the combination of posaconazole and sirolimus. Strain variability was noted among the same species. CONCLUSIONS: The clinical significance of these findings is unclear, but further studies are warranted given the potential for concomitant use of these agents in transplant patients treated for zygomycosis.

Rhodococcus equi lung infection in an allogeneic hematopoietic stem cell transplant recipient.

In this report, we describe a case of Rhodococcus equi lung infection diagnosed in an allogeneic hematopoietic stem cell transplant with oral graft-versus-host disease 3 months after stem cell infusion. The lung lesion persisted despite an approximate 3 months of vancomycin therapy, but then responded favorably to a combination of intravenous ertapenem at 1 g daily and oral rifampin at 600 mg daily for 1 month. An overview of Rhodococcus infection in transplant recipients is presented. This case and the discussed literature suggest that combination antibiotic therapy is warranted in patients with decreased humoral and cellular immunity.

Non-bacterial infections in allogeneic non-myeloablative stem cell transplant recipients.

Data on non-bacterial infections during allogeneic non-myeloablative hematopoietic stem cell transplantation (HSCT) are widely different. We evaluated data on 48 consecutive patients who received a conditioning regimen with fludarabine and cyclophosphamide (73%) or fludarabine and total body irradiation (27%) and then underwent allogeneic non-myeloablative HSCT. Cytomegalovirus (CMV) infection was common and occurred in 48% of patients; 3 patients developed CMV disease, and all survived. CMV reactivation was found to be common with both conditioning regimens in our patient population. Invasive aspergillosis occurred in 4 patients (8%) and 3 died. Other serious non-bacterial infections were uncommon. Review of the available literature on non-myeloablative HSCT suggests that the frequency and type of opportunistic infections vary considerably.

Micafungin: a new echinocandin.

Micafungin, a potent inhibitor of 1,3-beta-D-glucan synthase, has become the second available agent in the echinocandins class that is approved for use in clinical practice. This agent shares with caspofungin an identical spectrum of in vitro activity against Candida albicans, non-albicans species of Candida, and Aspergillus species, as well as several but not all pathogenic molds. If anything, its in vitro activity appears to be superior to that of caspofungin, although the clinical relevance of this observation is unclear. The clinical role of micafungin appears to be similar to that of caspofungin, although clinical data are still lacking at this stage, with initial approval only for treatment of esophageal candidiasis and prophylaxis in subjects with neutropenia. Pharmacokinetic and pharmacodynamic studies and reports of adverse effects and safety have reported similar but not identical results to those of other agents in the echinocandin class. Factors such as acquisition costs and the potential for resistance development may be more relevant to its widespread use than in vitro and in vivo data comparisons with caspofungin.

Effectiveness of amphotericin B lipid complex (ABLC) treatment in allogeneic hematopoietic cell transplant (HCT) recipients with invasive aspergillosis (IA).

A total of 85 allogeneic hematopoietic cell transplant (HCT) recipients with invasive aspergillosis treated with amphotericin B lipid complex (ABLC) were identified from the Collaborative Exchange of Antifungal Research (CLEAR) database. Of these patients, 78% (66/85) presented with pulmonary aspergillosis. Graft-versus-host disease (GVHD) was present in 24 of 85 patients. The response rate to ABLC was 31% (26/85) overall and 21% (5/24) in patients with GVHD. The overall response rate to first-line ABLC treatment was 41% (11/27). Four of nine (44%) patients with GVHD responded to first-line treatment with ABLC, while only one of 13 (8%) responded to ABLC as second-line therapy. Five of 18 (28%) and four of 14 (29%) patients, respectively, responded to sequential or concurrent treatment with ABLC and itraconazole. None of seven patients responded who continued receiving itraconazole after the start of ABLC therapy. At the end of ABLC therapy, serum creatinine had doubled in 12% of patients (10/85), and 2% (2/85) had developed a requirement for dialysis. These data suggest that ABLC, especially when administered as first-line therapy, can result in clinical response even in the most immunocompromised patients, that is, HCT recipients with GVHD, with minimal effects on renal function.

Antifungal resistance in Aspergillus.

There are currently few data on drug resistance in Aspergillus, but the recent development of susceptibility tests have made it possible, to some extent, to study resistance in azoles and polyenes. This paper reviews the limited data available. The increased incidence of aspergillosis and the introduction of newer drugs make it important to continue to monitor closely trends in antifungal drug resistance.

In-vitro activity of nikkomycin Z alone and in combination with polyenes, triazoles or echinocandins against Aspergillus fumigatus.

The in-vitro activity of nikkomycin Z was investigated in combination with polyenes, triazoles or echinocandins against 20 clinical isolates of Aspergillus fumigatus with the fractional inhibitory concentration index (FICI) method. The drug interactions were classified as synergic (FICI < or = 0.5), no interaction (FICI > 0.5, but FICI < or = 4) or antagonistic (FICI > 4). The fungicidal activity of nikkomycin Z alone and in combination with a representative echinocandin (caspofungin) or triazole (voriconazole) was also examined with time-kill experiments and fungal cell viability assays. Two-drug combinations of nikkomycin Z with amphotericin B (FICI 3.59 +/- 0.57), amphotericin B lipid complex (FICI 3.95 +/- 0.74), liposomal amphotericin B (FICI 3.62 +/- 0.98), itraconazole (FICI 2.0 +/- 0.0), voriconazole (FICI 1.07 +/- 0.37), posaconazole (FICI 2.20 +/- 0.44) or ravuconazole (FICI 1.76 +/- 0.44) showed no interactions, but the pairwise combination of nikkomycin Z with caspofungin (FICI 0.22 +/- 0.19) or micafungin (FICI 0.35 +/- 0.27) showed synergic activity against A. fumigatus. Time-kill studies and fungal cell viability assays showed that neither nikkomycin Z nor caspofungin alone possessed fungicidal activity against A. fumigatus, whereas a combination of these two drugs at concentrations > or = 2 mg/L (> or = 0.031 x the concentration of drug that produced no visible growth) killed germinated conidia within 24 h in a concentration-dependent manner. These data suggest that two-drug combinations of nikkomycin Z with echinocandins, but not with polyenes and triazoles, have a synergic effect against A. fumigatus.

Efficacy of voriconazole plus amphotericin B or micafungin in a guinea-pig model of invasive pulmonary aspergillosis.

The efficacy of voriconazole in combination with amphotericin B or micafungin was studied in a transiently neutropenic guinea-pig model of invasive pulmonary aspergillosis. Guinea-pigs treated with the antifungal drugs, alone or in two-drug combinations, had an improved survival rate and reduced fungal burden in the lungs compared to untreated control animals. The efficacy of monotherapy and combination therapy was similar; activity was neither enhanced nor reduced with the two-drug combinations. Further studies of efficacy, dosing and optimal regimens for antifungal combinations are warranted.

Caspofungin.

Available systemically effective antifungal agents for the treatment of invasive fungal infections are few. With the increasing recognition of a need for newer antifungal drugs, caspofungin has been introduced as the first member of a new class of compounds called echinocandins. This paper reviews the chemistry and mechanism of action of caspofungin, its activity in vitro and in animal models, and clinical pharmacokinetics,clinical efficacy and safety in patients.