Impact of enterococcal colonization and infection in solid organ transplantation recipients from the Swiss transplant cohort study.

BACKGROUND: The burden of enterococcal infections has increased over the last decades with vancomycin-resistant enterococci (VRE) being a major health problem. Solid organ transplantation is considered as a risk factor. However, little is known about the relevance of enterococci in solid organ transplantation recipients in areas with a low VRE prevalence. METHODS: We examined the epidemiology of enterococcal events in patients followed in the Swiss Transplant Cohort Study between May 2008 and September 2011 and analyzed risk factors for infection, aminopenicillin resistance, treatment, and outcome. RESULTS: Of the 1234 patients, 255 (20.7%) suffered from 392 enterococcal events (185 [47.2%] infections, 205 [52.3%] colonizations, and 2 events with missing clinical information). Only 2 isolates were VRE. The highest infection rates were found early after liver transplantation (0.24/person-year) consisting in 58.6% of Enterococcus faecium. The highest colonization rates were documented in lung transplant recipients (0.33/person-year), with 46.5% E. faecium. Age, prophylaxis with a betalactam antibiotic, and liver transplantation were significantly associated with infection. Previous antibiotic treatment, intensive care unit stay, and lung transplantation were associated with aminopenicillin resistance. Only 4/205 (2%) colonization events led to an infection. Adequate treatment did not affect microbiological clearance rates. Overall mortality was 8%; no deaths were attributable to enterococcal events. CONCLUSIONS: Enterococcal colonizations and infections are frequent in transplant recipients. Progression from colonization to infection is rare. Therefore, antibiotic treatment should be used restrictively in colonization. No increased mortality because of enterococcal infection was noted.

Impact of antiviral preventive strategies on the incidence and outcomes of cytomegalovirus disease in solid organ transplant recipients.

We assessed the impact of antiviral prophylaxis and preemptive therapy on the incidence and outcomes of cytomegalovirus (CMV) disease in a nationwide prospective cohort of solid organ transplant recipients. Risk factors associated with CMV disease and graft failure-free survival were analyzed using Cox regression models. One thousand two hundred thirty-nine patients transplanted from May 2008 until March 2011 were included; 466 (38%) patients received CMV prophylaxis and 522 (42%) patients were managed preemptively. Overall incidence of CMV disease was 6.05% and was linked to CMV serostatus (D+/R- vs. R+, hazard ratio [HR] 5.36 [95% CI 3.14-9.14], p < 0.001). No difference in the incidence of CMV disease was observed in patients receiving antiviral prophylaxis as compared to the preemptive approach (HR 1.16 [95% CI 0.63-2.17], p = 0.63). CMV disease was not associated with a lower graft failure-free survival (HR 1.27 [95% CI 0.64-2.53], p = 0.50). Nevertheless, patients followed by the preemptive approach had an inferior graft failure-free survival after a median of 1.05 years of follow-up (HR 1.63 [95% CI 1.01-2.64], p = 0.044). The incidence of CMV disease in this cohort was low and not influenced by the preventive strategy used. However, patients on CMV prophylaxis were more likely to be free from graft failure.

Treatment of cytomegalovirus infection or disease in solid organ transplant recipients with valganciclovir.

Valganciclovir (VGC) has proved efficacious and safe for the prophylaxis against cytomegalovirus (CMV) in high-risk transplant recipients and for the treatment of CMV retinitis in AIDS patients. We used VGC for the treatment of CMV infection (viremia without symptoms) or disease (CMV syndrome or tissue-invasive disease) in kidney, heart, and lung transplant recipients. Fourteen transplant recipients were treated: five for asymptomatic CMV infection and nine for CMV disease. VGC was administered in doses adjusted to renal function for 4 to 12 weeks (induction and maintenance therapy). Clinically, all nine patients with CMV disease responded to treatment. Microbiologically, treatment with VGC turned blood culture negative for CMV within 2 weeks in all patients and was associated with a > or =2 log decrease in blood CMV DNA within 3 weeks in 8 of 8 tested patients. With a follow-up of 6 months (n = 12 patients), asymptomatic recurrent CMV viremia was noted in five cases, and CMV syndrome noted in one case (all cases in the first 2 months after the end of treatment). VGC was clinically well tolerated in all patients; however, laboratory abnormalities occurred in three cases (mild increase in transaminases, thrombocytopenia, and pancytopenia). This preliminary experience strongly suggests that therapy with VGC is effective against CMV in organ transplant recipients; however, the exact duration of therapy remains to be determined: a longer course may be necessary to prevent early recurrence.

CMV retinitis recurs after stopping treatment in virological and immunological failures of potent antiretroviral therapy.

OBJECTIVES: To determine predictors of clinical relapse of cytomegalovirus (CMV) end-organ disease in a cohort of 17 HIV-infected patients with healed and treated CMV retinitis (CMVR) who responded to HAART with an increase in CD4 cell counts to above 70 cells/mm3 and discontinued CMV maintenance therapy (MT). DESIGN: Seventeen patients were monitored for reactivation of retinitis. The CD4 cell counts, HIV RNA and peripheral blood mononuclear cell (PBMC) lymphoproliferative assays to CMV at 3 month intervals were compared between patients with and without reactivation of CMVR. Positive lymphoproliferative responses were defined as a stimulation index of 3 or greater. RESULTS: Five out of 17 (29%) patients experienced a recurrence of CMVR a mean of 14.5 months after stopping CMV MT and between 8 days and 10 months after CD4 cell counts fell below 50 cells/mm3. Median CD4 cell counts and plasma HIV RNA at reactivation were 37 cells/mm3 and 5.3 log10 copies/ml. Three patients recurred at a previously active site of the retina, one had contralateral CMVR, and one a recurrence of retinitis and pancreatitis simultaneously. Mean lymphoproliferative responses to CMV were 2.4 in patients with reactivation versus 21.0 stimulation index (SI) in patients without reactivation (P= 0.01). A model incorporating four variables (CD4 cell counts and HIV RNA at maintenance discontinuation, highest CD4 cell count, nadir HIV RNA and median lymphoproliferative responses) identified correctly 88% of patients with and without reactivation. CONCLUSION: CMV disease recurs after virological and immunological failure of HAART if CD4 cell counts drop below 50. In this situation, anti-CMV agents should be resumed before clinical reactivation ensues, because of the risk of contralateral retinal involvement and systemic disease.

Sampling lymphoid tissue cells by ultrasound-guided fine needle aspiration of lymph nodes in HIV-infected patients. Swiss HIV Cohort Study.

OBJECTIVE: To establish the feasibility of using ultrasound-guided lymph node needle aspiration as a means to obtain lymphoid tissue cells for the determination of a series of immunologic and virologic measures in HIV-infected patients. DESIGN: First, a comparison of the characteristics of cell populations obtained by simultaneous needle aspiration and standard excisional biopsy in six patients. Second, use of lymph node needle aspiration to assess longitudinally T-cell subset changes in patients initiating highly effective antiretroviral treatment. METHODS: T-cell subsets (CD4 and CD8) and percentage Ki67+ cycling T cells were measured in lymph node cell populations harvested by ultrasound-guided aspiration or standard biopsy by flow cytometry. Cellular RNA content was assessed by a modification of the Roche Amplicor HIV-1 Monitor test. RESULTS: CD4 and CD8 T-cell percentage and HIV RNA cell content of lymph node cell suspensions obtained from the simultaneous performance of ultrasound-guided needle aspiration and excisional biopsy in the same patients were correlated (n = 6). Among the 87 aspiration sessions reported here, mononuclear cell suspensions were obtained in 100% of the sessions, in numbers ranging between 4x10(4) to 6.7x10(6) cells (median: 7x10(5)). This limited number of cells did not allow to perform all type of analyses in all patients. By prioritizing the cells for the determination of T-cell subsets and proliferation rate, this approach was instrumental for demonstrating the normalization of the T-cell subset ratio and the kinetic of normalization of proliferating rates of CD4 and CD8 T cells, as well as the decrease in HIV-1 viral load in the lymph node following HAART initiation. CONCLUSION: Ultrasound-guided aspiration appears to be a non-invasive and ad libitum, safe and repeatable procedure for the longitudinal monitoring of changes in lymph nodes.

The nef gene controls syncytium formation in primary human lymphocytes and macrophages infected by HIV type 1.

nef, the 3'-most open reading frame of HIV, has been reported to enhance HIV replication in various host cell types and to promote in vivo replication and pathogenesis. The mechanism underlying the increased in vivo viral replication is still unclear. We have examined the effect of a nef deletion on the infection of primary human CD4+ T lymphocytes and macrophages, using clones with nef and env sequences derived, respectively, from T cell- and macrophage-tropic viruses. The deletion of nef enhanced the formation of syncytia in CD4+ T lymphocytes infected with macrophage-tropic clones, despite a severalfold reduced viral production. No such enhancement of syncytium formation was observed in CD4+ T lymphocytes infected with a T cell line-tropic clone, but in this clone, the deletion of nef imparted a more severe replication defect. A similar increase in syncytium formation was observed in primary human macrophages infected with nef-deleted clones compared with wild-type counterparts, except under conditions in which the deletion of nef markedly reduced viral replication. We could not demonstrate an enhanced cell surface expression of HIV-1 envelope in lymphocytes infected with nef-deficient clones to explain the increased syncytium formation. In enhancing the HIV-1 cytopathic effect, the deletion of nef might curtail virus production by infected cells, and thus explain in part the reduced viral load observed in vivo in hosts infected with nef-deficient viruses.

Characterization of a macrophage-tropic HIV strain that does not alter macrophage cytokine production yet protects macrophages from superinfection by vesicular stomatitis virus.

Macrophages, unlike CD4+ T cells, can be productively infected by human immunodeficiency virus (HIV) without prior cellular activation. Cytopathic infection ensues without the induction of tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), or tissue factor genes. In detailed studies on TNF alpha, HIV infection did not affect the regulation of TNF alpha in response to bacterial lipopolysaccharide. In an effort to examine the interferon responsiveness of HIV-infected macrophages, the cells were challenged with vesicular stomatitis virus (VSV) with or without interferon pretreatment. Surprisingly, HIV-infected macrophages were completely resistant to VSV-induced lysis even in the absence of interferon; however, no interferon was detected in the supernatants of these infected cells. The resistance of HIV-infected macrophages to superinfection with VSV indicates a previously undescribed effect of HIV upon macrophage cellular metabolism.

Single tube competitive PCR for quantitation of CMV DNA in the blood of HIV+ and solid organ transplant patients.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in transplant and HIV-infected patients. However, CMV can also cause asymptomatic infection. An elevated blood viral load as assessed by various methods appears to be a predictor for symptomatic infections, and can be used to identify patients at the highest risk of developing CMV disease. We developed a single tube competitive quantitative PCR assay for CMV DNA, using as a competitor a plasmid carrying the target sequence for amplification with an internal deletion. The analysis of data from repeated extractions and amplifications of samples showed that the coefficient of variation of the assay was typically less than 20%. Clinical samples from 14 HIV-infected and 13 solid organ transplant patients were analyzed. Widely varying CMV DNA levels were found in leukocytes, with a positive correlation with the measure of infectivity in the leukocytes by quantitative culture on fibroblasts. The highest CMV DNA content in leukocytes was found in two patients with presumptive CMV disease. In HIV patients, the amount of DNA in leukocytes was much larger than in solid organ transplant recipients, when standardized for infectivity. Although based on a very limited number of patients, this observation probably points to a difference in the biology of CMV infection in these two categories of susceptible individuals. CMV DNA was also found in the plasma of some of the patients with a high CMV DNA leukocyte load. The present test should be useful for identifying patients at high risk of developing CMV disease, for monitoring therapeutic efficacy of antiviral regimens and to improve the understanding the pathogenesis of CMV infection.

Risk of transmission of human immunodeficiency virus infection during tympano-ossicular homograft: an experimental study.

It is generally agreed that middle ear reconstructive surgery performed with tympano-ossicular homografts produces superior functional results compared with prosthetic material, especially with respect to extrusion rate. The use of homografts, though, has been seriously hampered recently by the fear of transmission of human immunodeficiency virus (HIV) infection. In HIV-infected patients, the virus is primarily found in the cells of the lymphoid and monocytic lineage. The nature of the tissues in the eardrum and ossicles, mostly fibrous tissue and compact bone without marrow, suggests that little virus load should be found in homografts. Indeed, culturing minced homograft tissue from two HIV-infected donors with acquired immune deficiency syndrome (AIDS) in a sensitive culture system with PHA-stimulated lymphoblasts produced no virus. Before use, homografts undergo a fixation procedure in 5% formaldehyde and then are kept in a solution containing Cialit as a preservative. The authors therefore examined the capacity of formaldehyde and Cialit to reduce the infectivity of HIV in models of infected tissue as measured in vitro. The reduction of in vitro infectivity due to these treatments was at least 10(5)-fold and 10(2)-fold, respectively. Coupled with the low virus burden in tympano-ossicular tissue, our data suggests that the fixation procedure affords such a reduction in infectivity that the risk of HIV transmission, even from an HIV-infected donor, is vanishingly low.

Detection of CMV DNA in peripheral blood leukocytes as a long-range harbinger of CMV disease in HIV-infected patients. Swiss HIV Cohort Study.

The incidence of cytomegalovirus (CMV) end-organ disease can be reduced in AIDS patients by oral ganciclovir. However, the cost effectiveness of this prophylaxis is low. Targeting prophylaxis to patients with the highest risk of developing CMV disease might be useful. Several studies have shown the potential of various polymerase chain reaction (PCR) assays and of the antigenemia assay to identify the subset of patients with a definitely higher risk of developing CMV disease. We studied the CMV viral load using quantitative PCR in the leukocytes and plasma (or serum) of 28 patients in the four years before they experienced a CMV event. We observed rising CMV DNA copy numbers in the patients' leukocytes a year before the event. In contrast, plasma or serum copy numbers rose later and in fewer patients. In a control population of 21 profoundly immunodeficient patients (median CD4+ T cell count: 31/mm(3)) without history of CMV disease, only five had detectable CMV DNA in the leukocytes, three of whom had barely above-threshold levels. We suggest that, at the present time, leukocyte CMV DNA PCR might represent a sensitive test providing an early warning signal of increased risk of CMV disease. Performing it twice a year might identify patients at risk so that closer monitoring and targeted prophylaxis can be conducted.

Influenza transmission to recipient through lung transplantation.

We report a case of transmission of influenza A virus through lung transplantation. Given the prevalence of influenza during the yearly epidemic, the duration of viral excretion, and the risk of respiratory complications, the occurrence of such events needs to be considered during the influenza season.

Meningococcal disease in a kidney transplant recipient with mannose-binding lectin deficiency.

We describe the case of a kidney transplant recipient who developed meningococcemia, without meningeal signs, 2 months after transplantation. Plasma levels of complement components C3, C4, and CH 50 were within the normal range. However, using a method to screen for the functional activity of all 3 pathways of complement, no activation via the mannose-binding lectin (MBL) pathway could be detected (0%). A subsequent quantification of MBL pathway components revealed normal levels of MASP 2 but undetectable amounts of MBL. To our knowledge, this is the first report of meningococcal disease after organ transplantation in a patient with MBL deficiency.

[Increased risk of bacterial colonization of intravenous catheters covered with transparent adhesive polyurethane bandages, compared to classical gauze bandages]. / Risque accru de colonisation bactérienne des cathéters intraveineux recouverts de pansements transparents adhésifs en polyuréthane, comparé aux pansements classiques en gaze.

While transparent polyurethane dressings are increasingly used for the care of intravenous catheters, concern has recently been expressed regarding their microbiological safety. We have therefore compared the rate of intravenous catheter bacterial colonization after randomly assigning intensive care patients to transparent polyurethane (n = 21) or dry gauze (n = 20) dressings. Polyvinyl chloride catheters were inserted and maintained by the nurses. No antiseptic or antibiotic ointment was used. The two groups of patients were similar regarding risk factors for catheter colonization. Colonization rate was 48% (10/21) among patients with transparent dressings versus 10% (2/20) among patients with dry gauze dressings (p = 0.008). Colonizing bacterial species were Staphylococcus epidermidis (11 strains) and S. aureus (1 strain). No catheter-related bacteremia was observed. These data suggest that the colonization rate of intravenous catheters is increased by the use of polyurethane dressings, possibly increasing the risk of septic phlebitis and bacteremia.

Role of complement-derived and bacterial formylpeptide chemotactic factors in the in vivo migration of neutrophils in experimental Escherichia coli pyelonephritis in rats.

In experimental Escherichia coli pyelonephritis, the bacterial multiplication in the kidney parenchyma triggers a burst of neutrophil extravascular migration, as measured by the myeloperoxidase (MPO) activity in the kidney, a marker for tissue neutrophil infiltration. To test the mechanisms of in vivo neutrophil migration, pyelonephritis was surgically induced in rats that were then either complement-depleted with cobra venom factor (CVF), resulting in a profound hypocomplementemia for 72 h after inoculation, or treated with phenylbutazone (PB), a competitive antagonist of bacterial chemotactic formylpeptides. Compared to controls, CVF- and PB-treated animals killed when the neutrophil infiltration started (32 h) had a significantly reduced neutrophil infiltration, as measured by kidney MPO activity. This effect disappeared in animals killed 72 h after surgery, when neutrophil infiltration peaked. These data suggest that redundant chemotactic mechanisms triggered neutrophil migration. Inhibiting one of these mechanisms only transiently delayed neutrophil migration but did not affect the peak infiltration.

Failure of dexamethasone to prevent polymorphonuclear leukocyte infiltration during experimental acute exudative pyelonephritis and to reduce subsequent chronic scarring.

In experimental acute exudative pyelonephritis (AEP), a role for polymorphonuclear leukocyte (PMNL) infiltration in the pathogenesis of kidney scarring has been suggested indirectly. To directly quantitate PMNL infiltration during AEP, we developed an assay for measuring the content in the kidney of myeloperoxidase (MPO), an enzyme present in PMNLs and absent in kidney tissue. This assay was a specific and sensitive marker of the kidney PMNL content. We used this assay to measure in rats with AEP the effect of dexamethasone, administered in an attempt to mitigate the acute inflammatory response. Compared with saline, dexamethasone given during AEP strikingly reduced kidney swelling, measured by the kidney-weight increase, but failed to reduce PMNL infiltration, measured by the kidney MPO content. Despite reduced kidney swelling during AEP, dexamethasone treatment failed to prevent subsequent kidney scarring, an observation indicating that PMNLs play a role in the development of permanent kidney damage during AEP.

Discrepancies between MBC and actual killing of viridans group streptococci by cell-wall-active antibiotics.

We determined the MBC of amoxicillin and vancomycin, two antibiotics advocated for treatment and prophylaxis of bacterial endocarditis, for 24 strains of viridans group streptococci isolated from patients with endocarditis. We found that the MIC of amoxicillin for all strains was less than or equal to 0.25 micrograms/ml and the MBC was either low (less than 0.5 micrograms/ml) in 6 nontolerant strains or high (greater than 128 micrograms/ml) in 18 tolerant strains. The MIC of vancomycin for the 24 strains was less than or equal to 1 microgram/ml, and the MBC was either low (less than 1 microgram/ml) for 3 nontolerant strains or high (greater than 128 micrograms/ml) for 21 tolerant strains. In addition to the MBC, we determined the actual reduction of the viable bacterial counts in each tube dilution after 24 h of incubation. This determination was made by subtracting the number of colonies observed on the subculture plate from the number of bacteria contained in the initial inoculum. For both antibiotics we found that the maximal reduction in viable counts was achieved at or very close to the MIC and did not increase with increasing antibiotic concentrations (up to 128 micrograms/ml). As expected, the six strains for which the amoxicillin MBC was less than 0.5 micrograms/ml and the three strains for which the vancomycin MBC was less than 1 microgram/ml had a reduction of viable counts of more than 3 log10 (greater than 99.9% killing). In contrast, among the strains defined as tolerant to amoxicillin and vancomycin, there were wide variations in the actual reduction of bacterial counts, ranging from 3 log10 to less than 1 log10. Therefore our observations suggest that the reduction of viable streptococcal counts reflects more accurately the bactericidal effect of amoxicillin and vancomycin than does the MBC, which artificially divides the strains into sensitive or tolerant strains.

Influence of inflammation on the efficacy of antibiotic treatment of experimental pyelonephritis.

An acute exudative Escherichia coli pyelonephritis rat model was used to study the influence of progressive pyelonephritis on the efficacy of antibiotic treatment. In this model, transient ureteral obstruction after E. coli bladder inoculation induces early bacterial multiplication in the kidney parenchyma, and the bacterial counts peak by 48 h. The inflammatory response (assessed by the increase in kidney weight) is somewhat delayed, starting 36 h after inoculation and peaking by 72 h. Groups of rats received 4 doses over 48 h of saline, ceftriaxone (100 mg/kg), or ceftriaxone (100 mg/kg) plus gentamicin (4 mg/kg). These treatments were initiated 24, 36, 48, or 72 h after bladder inoculation. Antibiotic treatment started at 24 h was significantly more effective in reducing bacterial counts in the kidney parenchyma than at any later therapy onset. Only when started 24 h after inoculation was the synergistic combination of ceftriaxone plus gentamicin more effective in reducing bacterial counts than ceftriaxone alone. Ceftriaxone and ceftriaxone plus gentamicin regimens started at 24 h reduced significantly (by 42 and 55%, respectively) the incidence of acute exudative pyelonephritis when compared with the incidence in saline-treated controls. Early therapy onset (24 h) strikingly reduced the development of the inflammatory response. This reduction was less marked when antibiotic therapy was started at 36 h and no longer apparent when therapy onset was delayed up to 48 or 72 h. In conclusion, the efficacy of antibiotics in eradicating bacteria from the kidney parenchyma and in preventing acute exudative pyelonephritis was markedly hampered by the development of pyelonephritis.

Reduced intracellular growth of mycobacteria in human macrophages cultivated at physiologic oxygen pressure.

The growth of mycobacteria in human macrophages was examined at an ambient concentration of oxygen (5% CO2 and 95% air, corresponding to 20% O2 or 140 mm Hg PO2) and at a concentration corresponding to tissue levels (5% O2 and 5% CO2 in nitrogen balance, corresponding to 36 mm Hg PO2). Compared with the higher PO2 level, macrophages cultivated at lower PO2 level spread more widely and had an increased glycolytic and decreased oxidative metabolism. Upon PMA stimulation, they displayed a better preserved ability to produce superoxide anion and to respond to IFN-gamma priming by increased superoxide anion production. When infected with either Mycobacterium tuberculosis or Mycobacterium avium, macrophages cultured with the lower PO2 level permitted significantly less growth than those cultured at the higher PO2 level. From Day 0 to Day 7, M. tuberculosis grew an average of 0.39 and 1.17 log CFU in macrophages cultured at lower and higher PO2, respectively (p less than 0.0001). From Day 0 to Day 3 of infection, M. avium decreased in macrophages cultured at lower PO2 on average by 0.19 log CFU but grew by 0.34 log CFU in macrophages cultured at higher PO2 (p = 0.0001). Mycobacteria grew equally well in macrophage-free media at either PO2. Crude lymphokines, rIFN-gamma, or rTNF-alpha did not consistently affect the growth of mycobacteria in macrophages at either high or low oxygen conditions. In conclusion, mycobacteria displayed a reduced growth when cultivated in macrophages at a physiologic PO2 that did not reduce the growth of extracellular bacteria. This effect of PO2 on macrophage antimycobacterial power might explain the preferential localization of tuberculous lesions in body areas with high tissue PO2, such as the lung apex.