Resistance to recombinant human erythropoietin is a common condition in dialyzed patients with chronic kidney disease and is associated with more hospitalizations, increased mortality and frequent blood transfusions. The main cause of hyporesponsiveness to recombinant human erythropoietin in these patients is iron deficiency. However, a high proportion of patients does not respond to treatment, even to the use of intravenous iron, which indicates the presence of other important causes of resistance. In addition to the iron deficiency, the most common causes of resistance include inflammation, infection, malnutrition, inadequate dialysis, and hyperparathyroidism, although other factors may be associated. In the presence of adequate iron stores, other causes should be investigated and treated appropriately.

Despite the evidences showing the relevance of regulatory immune-mediated mechanisms to guarantee the stable graft function in renal transplanted patients, studies focusing on the immune response observed over a long-term period after renal transplantation are still limited. Several efforts have been done to establish novel biomarkers with relevant predictive values that could be used as prognostic laboratorial tools to monitor the complex network triggered through time after kidney transplantation. In this study, we have evaluated the pro-inflammatory and regulatory patterns of plasma cytokines in a group of 120 renal transplanted patients with stable graft function ranging from 1 to 160 months. Our data demonstrated an overall predominance of regulatory cytokines short-term after renal transplantation (1-24 months) with peaks of IL-4, IL-5 and IL-10. Moreover, a slight peak of TNF-α was observed 25-60 months after renal transplantation. Following a gap of stable cytokine profile (61-120 months), peaks of pro-inflammatory cytokines IL-8, IL-6, IL1β, TNF-α and IL-12 were observed later on (>120 months) after renal transplantation. Additionally, the categorical analysis of "low" or "high" cytokine producers re-enforce the occurrence of an overall regulatory status early-after stable renal graft function with a predominant pro-inflammatory pattern later on long-term renal transplantation. Taken together, our data suggest that IL-5 is a good biomarker associated with short-term stable renal function, whereas IL-12 seems to be a relevant pro-inflammatory element in long-term renal transplanted patients.

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A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10 6 leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.The diagnostic application of PCR presents challenges in response to which sophisticated modifications of the basic technique are continually being developed. For example, to ensure very-high-sensitivity qualitative diagnosis (one to five target genes in a clinical sample), nested PCR or a postamplification hybridization protocol has become routine (1,9). Where viral load is an issue, quantification is normally undertaken with external competitive standards and/or limiting dilution and multiple replicates (7,22). In general, such quantitative assays do not exhibit the levels of sensitivity of nested PCR or hybridization and are thus not appropriate where initial qualitative diagnosis is required. The plethora of alternative PCR strategies and the diverse requirements of the assay have led to a situation in which the clinical application of PCR has become both complex and costly.We have investigated whether a single, simple, carefully applied PCR assay might be able to substitute both for the use of nested PCR and postamplification hybridization for highsensitivity qualitative diagnosis and for complex quantification protocols for viral load assessment of the same infection. The model with which we chose to work is human cytomegalovirus (HCMV). High-sensitivity detection of HCMV is required for screening of blood donors, evaluation of amniotic fluid in anti-HCMV immunoglobulin M (IgM)-positive pregnant women, and analysis of spinal fluid samples. On the other hand, quantitative viral load assessment is required for monitoring of immunosuppress...

BackgroundParvovirus B19 presents tropism for human erythroid progenitor cells, causing chronic anemia in organ transplant recipients, due to their suppressed humoral and cellular responses. Diagnosis may be achieved through serological tests for detection of anti-B19 antibodies. However, renal transplant recipients are not routinely tested for parvovirus B19 infection, since there is scanty data or consensus on screening for B19 infection, as well as for treatment or preventive management of transplanted patients.Case presentationHerein we report a kidney transplant recipient, who was unresponsive to treatment of severe anemia, and presented hypocellular hematopoietic marrow, megaloblastosis and hypoplasia of erythroid lineage with larger cells with clear nuclei chromatin and eosinophilic nuclear inclusions. This patient was seropositive for Epstein-Barr and Cytomegalovirus infections and negative for anti-parvovirus B19 IgM and IgG antibodies, although symptoms were suggestive of parvoviruses infection. A qualitative polymerase chain reaction testing for B19 in serum sample revealed positive results for B19 virus DNA.ConclusionThis case report suggests that the diagnostic process for parvovirus B19 in renal transplant recipients should include a polymerase chain reaction assay to detect B19-DNA, since specific serological tests may be unreliable given their impaired humoral responses. These results also indicate the importance of considering parvovirus B19 infection in the differential diagnosis of persistent anemia in transplanted patients.