Very Early Cytomegalovirus Infection After Renal Transplantation: A Single-Center 20-Year Perspective.

BACKGROUND: Cytomegalovirus (CMV) infection risk in the first month after transplantation is felt to be minimal; however, the epidemiology has not been specifically investigated, particularly in the modern era of potent immunosuppressive regimens and universal CMV prophylaxis. OBJECTIVE: The aim of this study was to describe the incidence of and risk factors associated with CMV occurring less than 30 days after transplant and evaluate the effect of very early CMV on outcomes. METHODS: Retrospective, single-center study of adult renal transplant (RTX) recipients between January 1, 1994 and December 31, 2014. RESULTS: A total of 5225 patients who received a renal transplant in the study time period were reviewed for the presence of CMV infection occurring less than 30 days after transplant. Of these, only 14 patients demonstrated this finding for an overall incidence of 0.27%. Half of these patients were considered to be at heightened risk due to being a recipient of a non-primary transplant or on chronic immunosuppression. This left seven patients without known risk factors for very early CMV to evaluate. In this group, time from transplant to CMV infection was 13.5 ± 7 days. The majority (57.1%, n = 4) were high-risk serostatus (CMV D+/R-) and occurred in the valganciclovir era (71.4%, n = 5). Lymphocyte-depleting induction predominated (57.1%, n = 4). Average cold ischemic time (CIT) was 19.7 ± 7.7 hours. Three patients had post-operative complications, two required exploratory-laparotomy for hemorrhage. When evaluating outcomes, 43% (n = 3) had subsequent episodes of CMV infection, 28.6% (n = 2) developed rejection, and 28.6% (n = 2) died. Outcomes between patients with CMV infection less than 30 days and those with CMV infection more than 30 days after transplant were not significantly different. CONCLUSIONS: In our review of over 5000 kidney transplants, the incidence of CMV infection in the first 30 days after renal transplant is 0.2%. Notable common patient characteristics include hemorrhage requiring re-operation and prolonged CIT. Outcomes were similar to CMV occurring more than 30 days after transplant. This study should provide the clinician with some reassurance; despite potent immunosuppressive therapy, CMV infection in the first 30 days is unlikely.

Impact of ureteral stricture and treatment choice on long-term graft survival in kidney transplantation.

We aimed to evaluate the influence of urological complications occurring within the first year after kidney transplantation on long-term patient and graft outcomes, and sought to examine the impact of the management approach of ureteral strictures on long-term graft function. We collected data on urological complications occurring within the first year posttransplant. Graft survivals, patient survival, and rejection rates were compared between recipients with and without urological complications. Male gender of the recipient, delayed graft function, and donor age were found to be significant risk factors for urological complications after kidney transplantation (P < .05). Death censored graft survival analysis showed that only ureteral strictures had a negative impact on long-term graft survival (P = .0009) compared to other complications. Death censored graft survival was significantly shorter in kidney recipients managed initially with minimally invasive approach when compared to the recipients with no stricture (P = .001). However, graft survival was not statistically different in patients managed initially with open surgery (P = .47). Ureteral strictures following kidney transplantation appear to be strongly negatively correlated with long-term graft survival. Our analysis suggests that kidney recipients with ureteral stricture should be managed initially with open surgery, with better long-term graft survival.

Does DCD Donor Time-to-Death Affect Recipient Outcomes? Implications of Time-to-Death at a High-Volume Center in the United States.

For donation after circulatory death (DCD), many centers allow 1 h after treatment withdrawal to donor death for kidneys. Our center has consistently allowed 2 h. We hypothesized that waiting longer would be associated with worse outcome. A single-center, retrospective analysis of DCD kidneys transplanted between 2008 and 2013 as well as a nationwide survey of organ procurement organization DCD practices were conducted. We identified 296 DCD kidneys, of which 247 (83.4%) were transplanted and 49 (16.6%) were discarded. Of the 247 recipients, 225 (group 1; 91.1%) received kidneys with a time to death (TTD) of 0-1 h; 22 (group 2; 8.9%) received grafts with a TTD of 1-2 h. Five-year patient survival was 88.8% for group 1, and 83.9% for group 2 (p = 0.667); Graft survival was also similar, with 5-year survival of 74.1% for group 1, and 83.9% for group 2 (p = 0.507). The delayed graft function rate was the same in both groups (50.2% vs. 50.0%, p = 0.984). TTD was not predictive of graft failure. Nationally, the average maximum wait-time for DCD kidneys was 77.2 min. By waiting 2 h for DCD kidneys, we performed 9.8% more transplants without worse outcomes. Nationally, this practice would allow for hundreds of additional kidney transplants, annually.

Association Between Duration of Human Immunodeficiency Virus (HIV)-1 Viral Suppression Prior to Renal Transplantation and Acute Cellular Rejection.

Renal transplant has become an important option for human immunodeficiency virus (HIV)-infected patients with end-stage renal disease; however, these patients experience a high rate of acute cellular rejection (ACR). Guidelines do not currently exist for the optimal duration of viral suppression prior to transplantation. In a retrospective cohort analysis of 47 HIV-infected renal transplant recipients, we compared the rate of ACR between patients based on the length of time of viral suppression prior to transplantation. Of the patients who achieved viral suppression for >6 months but less than 2 years prior to transplantation (n = 15), 60% experienced ACR compared to 41% of patients suppressed at least 2 years or more (n = 32) prior to transplant (p = 0.21). Patients suppressed <2 years experienced ACR at 2.48 times the rate of those suppressed 2 years or longer. Induction immunosuppression, HLA mismatch and panel-reactive antibodies (PRAs) did not significantly differ between the two groups.

Diagnosis and Treatment of Pancreas Rejection.

Despite significant improvement in pancreas allograft survival, rejection of the pancreas remains a major clinical problem. In addition to cellular rejection of the pancreas, antibody-mediated rejection of the pancreas is now a well-described entity. The 2011 Banff update established comprehensive guidelines for the diagnosis of acute and chronic AMR. The pancreas biopsy is critical in order to accurately diagnose and treat pancreas rejection. Other modes of monitoring pancreas rejection we feel are neither sensitive nor specific enough. In this review, we examine recent advances in the diagnosis and treatment of pancreas rejection as well as describe practical diagnostic and treatment algorithms.

Sustained reduction of alloantibody secreting plasma cells and donor specific antibody with proteasome inhibition in mice.

The long-lived plasma cells, which develop after alloantigen sensitization, produce donor specific alloantibodies (DSAs) that generate a positive serum cross-match and preclude transplantation. Bortezomib, a proteasome inhibitor, is being investigated in clinical desensitization protocols, however preclinical studies in a transplant model are nonexistent. We hypothesized that sustained treatment with only a proteasome inhibitor would eliminate plasma cells and reduce DSA over time. Cardiac allografts were transplanted into murine recipients. Eight weeks after allograft rejection the proteasome inhibitor, bortezomib, was injected intravenously twice weekly for 60 days. Serum alloantibody responses were assayed using flow cross-match. Total and alloreactive plasma cell numbers were enumerated using flow cytometry and ELISPOT. All recipients of cardiac allografts rejected their graft promptly within 16 days and demonstrated alloantibody by flow cross-match. DSA was sustained in the control mice while mice treated with bortezomib had sustained elimination of DSA and a marked reduction in plasma cell population. Also, bortezomib was associated with an increased level of BLyS. Within a murine model, proteasome inhibition can eliminate alloantibody secreting plasma cells, and reduce alloantibody. Cessation of bortezomib is not associated with return of DSA.

Decentralization of CD4 testing in resource-limited settings: 7 years of experience in six African countries.

Improving access to CD4 testing in resource-limited settings can be achieved through both centralized and decentralized testing networks. Decentralized testing models are more suitable for countries where the HIV epidemic affects a large portion of rural populations. Timely access to accurate CD4 results is crucial at the primary level of the health system. For the past 7 years, the Institute of Human Virology of the University of Maryland School of Medicine has implemented a flexible and sustainable three-phase model: (1) site assessment and improvement, (2) appropriate technology selection with capacity building through practical training and laboratory mentoring, and (3) quality management system strengthening and monitoring, to support accessibility to reliable CD4 counting at the point of service. CD4 testing capacity was established in 122 of 229 (53%) laboratories supported in Nigeria, Uganda, Kenya, Zambia, Tanzania, and Rwanda. Among those in rural settings, 46% (69/151) had CD4 testing available at site level, with a functioning flow cytometer installed at 28% (8/29) and 50% (61/122) of level 1 and level 2 sites, respectively. To strengthen local capacity, a total of 1,152 laboratory technicians were trained through 188 training sessions provided both on-site and at central locations. The overall quality of CD4 total testing procedure was assessed at 76% (92/121) of the laboratories, with 25% (23/92), 34% (31/92), and 33% (30/92) of them reporting excellent, good, and satisfactory performance. Balancing country-specific factors with the location of the clinic, number of patients, and the expected workload, was crucial in adapting this flexible model for decentralizing CD4 testing. The close collaboration with local governments and private vendors was key to successfully expanding access to CD4 testing within the framework of HIV care and treatment programs and for the sustainability of medical laboratories in resource-limited settings.

Situational analysis of varying models of adherence support and loss to follow up rates; findings from 27 treatment facilities in eight resource limited countries.

OBJECTIVES: Large-scale provision of ART in the absence of viral load monitoring, resistance testing, and limited second-line treatment options places adherence support as a vital therapeutic intervention. We aimed to compare patient loss to follow up rates with the degree of adherence support through a retrospective review of patients enrolled in the AIDSRelief program between August 2004 and June 2005. METHODS: Loss to follow up data were analysed and programs were categorised into one of four tiered levels of adherence support models: Tier I, II, III, and IV which increase from lowest to highest support. Bivariate and t-test analyses were used to test for significant differences between the models. RESULTS: 13,391 patients at 27 treatment facilities from six African and two Caribbean countries began antiretroviral therapy within the first year of the AIDSRelief program. The mean loss to follow up within the first year was 7.5%. Eight facilities were Tier I, three (Tier II), nine (Tier III), and seven (Tier IV). Facilities in Tier I had a loss to follow up rate of 14%, Tier II (10%), Tier III (5%), and Tier IV (1%). The proportion of loss to follow up for Tier I and Tier III were significantly different from each other (P < 0.02), as were Tier I and Tier IV (P < 0.006). There were differences between Tier II and Tier IV (P < 0.009) as well as Tier III and Tier IV (P < 0.017). CONCLUSION: These data strongly support the use of proactive adherence support programs, beyond routine patient counselling and defaulter tracking to support the'public health approach'to ART.

Bacterial DNA uptake sequences can accumulate by molecular drive alone.

Uptake signal sequences are DNA motifs that promote DNA uptake by competent bacteria in the family Pasteurellaceae and the genus Neisseria. The genomes of these bacteria contain many copies of their canonical uptake sequence (often >100-fold overrepresentation), so the bias of the uptake machinery causes cells to prefer DNA derived from close relatives over DNA from other sources. However, the molecular and evolutionary forces responsible for the abundance of uptake sequences in these genomes are not well understood, and their presence is not easily explained by any of the current models of the evolution of competence. Here we describe use of a computer simulation model to thoroughly evaluate the simplest explanation for uptake sequences, that they accumulate in genomes by a form of molecular drive generated by biased DNA uptake and evolutionarily neutral (i.e., unselected) recombination. In parallel we used an unbiased search algorithm to characterize genomic uptake sequences and DNA uptake assays to refine the Haemophilus influenzae uptake specificity. These analyses showed that biased uptake and neutral recombination are sufficient to drive uptake sequences to high densities, with the spacings, stabilities, and strong consensus typical of uptake sequences in real genomes. This result greatly simplifies testing of hypotheses about the benefits of DNA uptake, because it explains how genomes could have passively accumulated sequences matching the bias of their uptake machineries.

Coevolution of DNA uptake sequences and bacterial proteomes.

Dramatic examples of repeated sequences occur in the genomes of some naturally competent bacteria, which contain hundreds or thousands of copies of short motifs called DNA uptake signal sequences. Here, we analyze the evolutionary interactions between coding-region uptake sequences and the proteomes of Haemophilus influenzae, Actinobacillus pleuropneumoniae, and Neisseria meningitidis. In all three genomes, uptake sequence accumulation in coding sequences has approximately doubled the frequencies of those tripeptides specified by each species' uptake sequence. The presence of uptake sequences in particular reading frames correlated most strongly with the use of preferred codons at degenerately coded positions, but the density of uptake sequences correlated only poorly with protein functional category. Genes lacking homologs in related genomes also lacked uptake sequences, strengthening the evidence that uptake sequences do not drive lateral gene transfer between distant relatives but instead accumulate after genes have been transferred. Comparison of the uptake sequence-encoded peptides of H. influenzae and N. meningitidis proteins with their homologs from related bacteria without uptake sequences indicated that uptake sequences were also preferentially located in poorly conserved genes and at poorly conserved amino acids. With few exceptions, amino acids at positions encoded by uptake sequences were as well conserved as other amino acids, suggesting that extant uptake sequences impose little or no constraint on coding for protein function. However, this state is likely to be achieved at a substantial cost because of the selective deaths required to eliminate maladaptive mutations that improve uptake sequences.

Expansion of HIV-1 Screening and Anti-Retroviral Treatment Programs in a Resource-Poor Setting: Results from a Faith-Based Organization in Jos; Plateau State; Nigeria

Background: Until recently; availability of anti-retroviral therapy (ART) in Nigeria has been limited to government and university based programs. Through the United States' President's Emergency Plan For AIDS Relief (PEPFAR); additional funding has become available for the treatment of HIV-positive patients. Objective: To report the expansion of HIV-1 screening; enrollment in an ART program; and treatment outcomes over twelve months among HIV-positive patients at a nonprofit; non-governmental faith-based clinic providing free and holistic care in Jos City; Plateau State; Nigeria. Design: This was a retrospective analysis of HIV-1 screening and ART received by patients at Faith Alive Foundation Hospital (FAFH). From January through December 2005; voluntary counseling and testing (VCT) was freely available to all patients who requested it. Also beginning in December 2004; HIV-1 infected patients were enrolled in an expanded HIV/AIDS program at FAFH; where patients in clinical stages 3 or 4 based on World Health Organiza- tion (WHO) clinical classification; or had a CD4+ cell count of less than 200/ ml; were initiated on a simplified highly active ART (HAART) regimen through PEPFAR funding. Data from the first six quarters of the FAFH-PEPFAR ART program are presented here. Additionally; analysis of 645 patients who have received ART for one year; including changes in CD4+ cell count from baseline is shown. Results: In 2005; a total of 7672 persons received VCT and 3869 (50.4) HIV-1 positive results were found. From October 2004 to March 2006; the total number of patients enrolled in the FAFH HIV/AIDS program rose from 1330 to 5946 people. Over the same period; the number of patients who received ART increased from 302 to 1667. A majority of patients received an oral ART regimen consisting of generic nevirapine; lamivudine; and stavudine. The number of patients initiated on ART each quarter ranged from 57-578; and the number of deaths of patients on ART was between 12 and 21 people per quarter. Analysis of 645 patients initiated on ART during the first quarter of the FAFH-PEPFAR ART program revealed that the median CD4+ cell count at baseline was 106/ml and the median increase was 173/ml (median time of follow-up; 7.0 months). Conclusions: Comprehensive HIV/AIDS care has been significantly expanded at FAFH; a free and holistic medical center in Jos; Nigeria. Cumulative data from the first year of this PEPFAR-funded ART program indicated that promising outcomes are achievable through delivery of care at this faith-based medical center

Evolutionary stability of DNA uptake signal sequences in the Pasteurellaceae.

The DNA-uptake signal sequence (USS) of the bacterium Haemophilus influenzae is highly over-represented in its genome (1,471 copies of the core sequence AAGTGCGGT), and DNA fragments containing USS are preferentially taken up by competent cells. Because this bias favors uptake of conspecific DNA, USSs are often considered a kind of mate recognition system in bacteria, acting as species-specific barriers against uptake of unrelated DNA. However, the H. influenzae USS is highly over-represented in the genomes of three otherwise-divergent Pasteurellaceae species (Pasteurella multocida, Haemophilus somnus, and Actinobacillus actinomycetemcomitans, 927, 1,205, and 1,760 copies, respectively), suggesting that USSs do not always limit exchange. USSs in all these genomes are mainly in coding regions and show no orientation bias around the chromosome, weakening proposed USS functions in transcription termination and chromosome replication. Alignment of homologous genes was used to determine evolutionary relationships between individual USSs. Most H. influenzae USSs were found to have perfect or imperfect homologs (USS at the same location) in at least one other species, and most USSs in the other species had perfect or imperfect homologs in H. influenzae. These homologies suggest that the use of a common USS is due to inheritance of the USS-based uptake system from a common ancestor of the Pasteurellaceae, and it indicates that individual USSs can be evolutionarily stable elements of their genomes. The pattern is consistent with a molecular drive model of USS evolution, with new USSs arising by mutation and preferentially spread to new genomes by the biased DNA-uptake system.

Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV beta-chemokines: an approach to suppress R5 strains of HIV-1.

Propagation of R5 strains of HIV-1 on CD4 lymphocytes and macrophages requires expression of the CCR5 coreceptor on the cell surface. Individuals lacking CCR5 (CCR5 Delta 32 homozygous genotype) are phenotypically normal and resistant to infection with HIV-1. CCR5 expression on lymphocytes depends on signaling through the IL-2 receptor. By FACS analysis we demonstrate that rapamycin (RAPA), a drug that disrupts IL-2 receptor signaling, reduces CCR5 surface expression on T cells at concentrations as low as 1 nM. In addition, lower concentrations of RAPA (0.01 nM) were sufficient to reduce CCR5 surface expression on maturing monocytes. PCR analysis on peripheral blood mononuclear cells (PBMCs) showed that RAPA interfered with CCR5 expression at the transcriptional level. Reduced expression of CCR5 on PBMCs cultured in the presence of RAPA was associated with increased extracellular levels of macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta. In infectivity assays, RAPA suppressed the replication of R5 strains of HIV-1 both in PBMC and macrophage cultures. In total PBMC cultures, RAPA-mediated inhibition of CCR5-using strains of HIV-1 occurred at 0.01 nM, a concentration of drug that is approximately 103 times lower than therapeutic through levels of drug in renal transplant recipients. In addition, RAPA enhanced the antiviral activity of the CCR5 antagonist TAK-779. These results suggest that low concentrations of RAPA may have a role in both the treatment and prevention of HIV-1 infection.

Differential human immunodeficiency virus-suppressive activity of reverse transcription inhibitors in resting and activated peripheral blood lymphocytes: implications for therapy.

OBJECTIVES: Because recent evidence indicates that human immunodeficiency virus type 1 (HIV-1) propagates in resting T lymphocytes in vivo, we wanted to evaluate the antiviral effects exerted by currently used nucleoside (NRTI) and non-nucleoside analog reverse transcription inhibitors in resting lymphocytes, and compare those effects to the ones obtained in activated lymphocytes. METHODS: Tissue culture antiviral assays in which target cells are lymphocytes present in a resting or activated state. Virus replication was measured by a reverse transcription (RT) assay. Cell viability was evaluated using a commercial 3-(4k5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: In vitro results obtained with concentrations of zidovudine and stavudine equivalent to drug levels found in plasma, showed more than 99% HIV-1 inhibition in activated lymphocytes but less than 50% virus inhibition in resting lymphocytes. Conversely, plasma levels of didanosine-inhibited HIV-1 by approximately 50% and 98% in activated and resting lymphocytes, respectively. Plasma level concentration of zalcitabine, lamivudine, and abacavir inhibited viral replication by more than 90% in both resting and activated cells. CONCLUSIONS: These data demonstrate that specific NRTI antiretroviral agents have different activity against HIV RT, depending on the state of cell cycle of the infected cell. We suggest that the replication of HIV-1 in resting lymphocytes should be taken into account in the design of future clinical trials, as well as treatment antiretroviral regimens. Selection of combination RTIs so that they provide antiretroviral activity in both resting and activated lymphocytes may be a way to minimize treatment failure and the emergence of drug-resistant variants.

Do bacteria have sex?

Do bacteria have genes for genetic exchange? The idea that the bacterial processes that cause genetic exchange exist because of natural selection for this process is shared by almost all microbiologists and population geneticists. However, this assumption has been perpetuated by generations of biology, microbiology and genetics textbooks without ever being critically examined.

Competence development by Haemophilus influenzae is regulated by the availability of nucleic acid precursors.

DNA uptake by naturally competent bacteria provides cells with both genetic information and nucleotides. In Haemophilus influenzae, competence development requires both cAMP and an unidentified signal arising under starvation conditions. To investigate this signal, competence induction was examined in media supplemented with nucleic acid precursors. The addition of physiological levels of AMP and GMP reduced competence 200-fold and prevented the normal competence-induced transcription of the essential competence genes comA and rec-2. The rich medium normally used for growth allows only limited competence. Capillary electrophoresis revealed only a subinhibitory amount of AMP and no detectable GMP, and the addition of AMP or GMP to this medium also reduced competence 20- to 100-fold. Neither a functional stringent response system nor a functional phosphoenolpyruvate:glycose phosphotransferase system (PTS) was found to be required for purine-mediated repression. Added cAMP partially restored both transcription of competence genes and competence development, suggesting that purines may reduce the response to cAMP. Potential binding sites for the PurR repressor were identified in several competence genes, suggesting that competence is part of the PUR regulon. These observations are consistent with models of competence regulation, in which depleted purine pools signal the need for nucleotides, and support the hypothesis that competence evolved primarily for nucleotide acquisition.

Oxymonads are closely related to the excavate taxon Trimastix.

Despite intensive study in recent years, large-scale eukaryote phylogeny remains poorly resolved. This is particularly problematic among the groups considered to be potential early branches. In many recent systematic schemes for early eukaryotic evolution, the amitochondriate protists oxymonads and Trimastix have figured prominently, having been suggested as members of many of the putative deep-branching higher taxa. However, they have never before been proposed as close relatives of each other. We amplified, cloned, and sequenced small-subunit ribosomal RNA genes from the oxymonad Pyrsonympha and from several Trimastix isolates. Rigorous phylogenetic analyses indicate that these two protist groups are sister taxa and are not clearly related to any currently established eukaryotic lineages. This surprising result has important implications for our understanding of cellular evolution and high-level eukaryotic phylogeny. Given that Trimastix contains small, electron-dense bodies strongly suspected to be derived mitochondria, this study constitutes the best evidence to date that oxymonads are not primitively amitochondriate. Instead, Trimastix and oxymonads may be useful organisms for investigations into the evolution of the secondary amitochondriate condition. All higher taxa involving either oxymonads or Trimastix may require modification or abandonment. Affected groups include four contemporary taxa given the rank of phylum (Metamonada, Loukozoa, Trichozoa, Percolozoa), and the informal excavate taxa. A new "phylum-level" taxon may be warranted for oxymonads and Trimastix.

Synergistic inhibition of HIV-1 in activated and resting peripheral blood mononuclear cells, monocyte-derived macrophages, and selected drug-resistant isolates with nucleoside analogues combined with a natural product, resveratrol.

Resveratrol (trans-3,5,4;-trihydroxystilbene) is a phytoalexin present in grapes, wine, and certain plants, which has recently been reported to possess properties that may protect against atherosclerosis, certain cancers, and inflammation. We now report that resveratrol (RV) synergistically enhances the anti-HIV-1 activity of the nucleoside analogues zidovudine (AZT), zalcitabine (ddC), and didanosine (ddI). RV at 10 microM was not toxic to cells, and by itself reduced viral replication by 20% to 30%. In phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs) infected with HTLV-IIIB, 10 microM RV reduced the 90 % inhibitory concentration (IC90) of AZT, ddC, and ddI by 3.5-, 5.5-, and 17.8-fold, respectively. Similar antiviral activity was demonstrated when ddI was combined with 5 or 10 mM RV in PBMCs infected with clinical isolates of HIV-1. The addition of RV resulted in a >10-fold augmentation of ddI-antiviral activity in infected monocyte-derived macrophages (MDMs). In a resting cell model of T lymphocytes which were infected with HTLV-IIIB, RV plus ddI in combination, but not individually, suppressed establishment of a productive viral infection. In addition, RV plus ddI markedly inhibited the replication of four ddI-resistant viral isolates, three of which presented mutations in the RT gene conferring RT-multidrug resistance. Finally, when compared with hydroxyurea (HU), both 100 mM HU and 10 mM RV showed similar enhancement of ddI-antiviral suppressive activity. However, RV was shown to have less of a cellular antiproliferative effect than HU.