Use of Laboratory Assays To Predict Cytomegalovirus Disease in Renal Transplant Recipients

Eight laboratory assays, viz., the pp65 direct antigenemia test, a quantitative cytomegalovirus (CMV)-specific immunoglobulin G (IgG) assay (Biomerieux VIDAS), a CMV-specific IgM assay (Biomerieux VIDAS), the Hybrid Capture system (Murex), an in-house PCR with plasma (P-PCR) and leukocytes (L-PCR), and a commercial PCR (Roche AMPLICOR) with plasma (P-AMP) and leukocytes (L-AMP), were compared for their abilities to predict CMV disease before the onset of illness in a prospective study of 37 renal transplant recipients. By using an expanded criterion for active infection (two or more of the markers positive) and a clinical definition of disease, 22 (59%) patients were identified as having active CMV infection and 13 (35%) were identified as having CMV disease. Of the 13 CMV-seronegative recipients who received seropositive kidneys (R− group), 8 had active infection and disease. All assays were 100% specific and 100% predictive of CMV disease in the R− group. The leukocyte PCRs (L-PCR and L-AMP) were the most sensitive assays, had positive results an average of between 8 and 13 days before the onset of illness, and were the assays of choice. The performance of the assays was less satisfactory for the 24 patients who were CMV seropositive before transplantation (R+ group). A negative result was more useful for this group. Overall, P-AMP had the best results, and it could be the assay of choice for monitoring R+ patients. The non-PCR-based methods generally had high specificities but often gave late positive results and were not sensitive enough for use as prediction tools for either group of patients.     

Evaluation of New Quantitative Assays for Diagnosis and Monitoring of Cytomegalovirus Disease in Human Immunodeficiency Virus-Positive Patients

Cobas Amplicor CMV Monitor (CMM) and Quantiplex CMV bDNA 2.0 (CMV bDNA 2.0), two new standardized and quantitative assays for the detection of cytomegalovirus (CMV) DNA in plasma and peripheral blood leukocytes (PBLs), respectively, were compared to the CMV viremia assay, pp65 antigenemia assay, and the Amplicor CMV test (P-AMP). The CMV loads were measured in 384 samples from 58 human immunodeficiency virus (HIV) type 1-infected, CMV-seropositive subjects, including 13 with symptomatic CMV disease. The assays were highly concordant (agreement, 0.88 to 0.97) except when the CMV load was low. Quantitative results for plasma and PBLs were significantly correlated (Spearman rho = 0.92). For PBLs, positive results were obtained 125 days before symptomatic CMV disease by CMV bDNA 2.0 and 124 days by pp65 antigenemia assay, whereas they were obtained 46 days before symptomatic CMV disease by CMM and P-AMP. At the time of CMV disease diagnosis, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 97.8, 92.3, and 97.8%, respectively, whereas they were 92.3, 93.3, 80, and 97. 8%, respectively, for the pp65 antigenemia assay; 84.6, 100, 100, and 95.7%, respectively, for CMM; and 76.9, 100, 100, and 93.8%, respectively, for P-AMP. Considering the entire follow-up, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 73.3, 52.1, and 97.1%, respectively, whereas they were 100, 55.5, 39.4, and 100%, respectively, for the pp65 antigenemia assay; 92.3, 86.7, 66.7, and 97.5%, respectively, for CMM; and 84.6, 91.1, 73.3, and 95.3%, respectively, for P-AMP. Detection of CMV in plasma is technically easy and, despite its later positivity (i.e., later than in PBLs), can provide enough information sufficiently early so that HIV-infected patients can be effectively treated. In addition, these standardized quantitative assays accurately monitor the efficacy of anti-CMV treatment.     

Evaluation of PCR Primers for Early Diagnosis of Cytomegalovirus Infection following Liver Transplantation

The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the HindIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to EcoRI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% (HindIII-X) to 20% (MIE gene); however, specificity was inversely related (HindIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When HindIII-X and EcoRI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the HindIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.     

Monitoring of Cytomegalovirus Viral Loads by Two Molecular Assays in Whole-Blood and Plasma Samples from Hematopoietic Stem Cell Transplant Recipients

Cytomegalovirus (CMV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Roche Cobas AmpliPrep/Cobas TaqMan CMV test (Cobas CMV) has recently been cleared by the FDA for the monitoring of CMV viral loads in plasma samples from transplant patients. In this study, we compare and correlate the viral loads obtained by a laboratory-developed test (LC CMV) (using Roche analyte-specific reagents [ASR] on the LightCycler 2.0) on whole-blood specimens with those obtained on corresponding plasma and whole-blood specimens by the Cobas CMV assay. Testing was performed on 773 archived patient specimens. The strength of the agreement was good for the two assays performed on whole blood (κ = 0.6; 95% confidence interval [CI], 0.51 to 0.7) and moderate when the tests were performed on different sample types (κ = 0.54; 95% CI, 0.47 to 0.62 for the LC CMV whole blood [WB] assay versus Cobas plasma [PL], and κ = 0.57; 95% CI, 0.5 to 0.65 for the Cobas WB assay versus Cobas PL), although the difference was not statistically significant. Using a combination gold standard (i.e., a true positive was a specimen that was positive by two or more methods), the sensitivity and specificity of the assays were 78.8% and 99.3% for the LC CMV assay, 85.2% and 98.1% for the Cobas CMV WB assay, and 100% and 90.5% for Cobas CMV PL assay, respectively. A comparison of the CMV viral load trends in both plasma and whole blood from a few patients with multiple positive successive samples showed similar slopes, with differences in the slope ranging from 0.01 to 0.22. However, the absolute value for individual viral load differed markedly with whole-blood viral loads, being on average 0.5- to 1.22-log higher than those in plasma. The Cobas CMV assay provides a valid option for the monitoring of viral loads in transplant patients. Due to its increased sensitivity, the detection of CMV DNA in patients with low viral loads (i.e., those below limit of quantification [LOQ]) is increased with the Cobas CMV assay in plasma specimens. Longitudinal prospective studies will be needed to examine the clinical significance of these low-level viral loads.     

Cellular Immune Response to Cytomegalovirus Infection After Renal Transplantation

A prospective study of 15 patients who received renal transplants defined the effect of renal transplantation on the cellular immune response to cytomegalovirus infection. Of 15 patients, 14 developed cytomegalovirus infection, usually in the first 2 months after transplantation, and all infections were accompanied by a normal humoral immune response. After the initiation of immunosuppressive therapy and transplantation, there was a general depression of lymphocyte transformation, as reflected in the response to phytohemagglutinin, accompanied by a specific defect in cellular immunity, as indicated by lymphocyte transformation to cytomegalovirus antigen. Eleven patients had cellular immunity to cytomegalovirus before transplantation, and all of these became negative in the first month after transplantation. In subsequent months, only 6 of the 14 study patients with cytomegalovirus infection developed specific cellular immune responses to cytomegalovirus. This occurred most often in patients who had severe febrile illnesses in association with infection. The specific cellular immune response which developed in the posttransplant period did not persist in three of the patients. This study demonstrates the dissociation of the humoral and cellular immune response to cytomegalovirus infection in renal transplant patients and indicates the importance of the loss of cellular immunity in the appearance of infection. Previously infected patients lost their cell-mediated immunity and had reactivation infections despite the presence of serum antibody.     

肾移植术后感染性腹泻的诊疗分析

肾移植是挽救晚期慢性肾衰竭患者的最佳方式,与长期透析比较可使患者获得更长久的生存率,但由于术后需要长期服用免疫抑制剂和相对较长时间的抗生素,极易容易引起肠道菌群紊乱和肠道感染,导致腹泻的发生。本文着重回顾了有关肾移植受者感染性腹泻的现有文献,对肾移植后感染性腹泻的研究进展进行了总结,并希望为临床能寻找到高效可靠的移植后感染性腹泻的评估方法提供一定的思路。     

监测肾移植患者口服环孢素A血药浓度的三点法

环孢素(cyclospoRINE A,CsA)是一种有效的免疫抑制剂,广泛用于肾器官移植。由于它对肾有毒性反应和药代动力学参数个体差异大,因此监测和预测血药浓度对维持病人最佳免疫抑制水平很重要。 本文对十七例异体肾移植病人用:三点法“所构成血药浓度随时间变化的数学模型进行验证,为合理地调整给药剂量提供了依据。     

Molecular Fine-Specificity Analysis of Antibody Responses to Human Cytomegalovirus and Design of Novel Synthetic-Peptide-Based Serodiagnostic Assays

To identify single immunodominant marker proteins which can replace complex virion antigen in serodiagnostic assays, we investigated in detail the molecular fine specificity of antibody responses in different individuals with latent or active human cytomegalovirus (HCMV) infection. An overview of the HCMV proteins recognized by human antibodies was obtained by immunoblotting. For selected immunodominant proteins the epitope fine specificity of the antibody response was determined by a peptide-scanning enzyme-linked immunosorbent assay (ELISA). Epitope clusters were synthesized as combination peptides and were used for further serologic analysis of immunoglobulin M (IgM) and IgG reactivities with panels of sera from different groups of patients in comparison to those with cytomegalovirus (CMV) virion antigen. Several serum samples had significantly higher reactivities with peptides than with the CMV virion antigen. However, individual serum samples occasionally recognized diverse peptide epitopes, stressing the importance of using combinations of peptides in serologic assays. From these studies we were able to define a specific combination of peptides derived from pp52 (UL44) and pp150 (UL32) for the specific and highly sensitive early detection of HCMV IgM, whereas a combination of peptides from pp150 (UL32), gB (UL55), and pp28 (UL99) was selected to give optimal and specific reactivity with HCMV IgG. On the basis of the results obtained with these peptide combinations, new, highly specific serodiagnostic assays were constructed. These assays had sensitivities of 98.9 and 96.4% for IgG and IgM, respectively, in comparison with the results obtained with the “gold standard,” the virion antigen-based ELISA. From the results of this study we conclude that specific combinations of highly defined synthetic peptides can replace complex HCMV virion extracts used in current serodiagnostics and may add to further standardization of HCMV serology.     

Spontaneous Bladder Rupture and Cytomegalovirus Infection Complicating Renal Transplantation: Cause or Coincidence?

The high incidence of surgical complications following renal transplantation is well known. Urologic complications, however, present some of the most challenging problems to the transplant surgeon. The authors present here a detailed case report of spontaneous (delayed) bladder rupture (SDBR) which occurred 90 days after kidney transplantation in a recipient with cytomegalovirus infection (CMV). Urinary catheter drainage is recommended in preference to surgical intervention for the successful correction of SDBR. It is postulated further that, despite a negative bladder biopsy, CMV may have infiltrated the bladder and contributed to this “spontaneous” bladder wall rupture.     

Improvements in Methods for Disease Diagnosis and Monitoring

Biomedical Engineering - A review is made of the main scientific studies presented in the section Methods and Means of Diagnosis and Treatment of Various Diseases of the XIVth International...     

Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies

Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log₁₀ copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated.     

Quantitative PCR Is Faster,More Objective,and More Reliable Than Immunohistochemistry for the Diagnosis of Cytomegalovirus Gastrointestinal Disease in Allogeneic Stem Cell Transplantation

Diagnosis of gastrointestinal (GI) cytomegalovirus (CMV) disease relies on the presence of GI symptoms and detection of CMV, mainly by immunohistochemistry (IHC), in GI biopsy specimens. Thus, in a symptomatic patient, a positive CMV-IHC result is accepted as a diagnosis of CMV disease. However, a positive CMV-PCR in GI tissue is considered “possible” CMV disease. Therefore, it would be very useful if, in practice, both techniques showed equal sensitivity and reliability. This is because PCR has many practical advantages over IHC for detecting CMV. The aim of this study was to compare quantitative PCR with IHC for the diagnosis of GI CMV disease. A total of 186 endoscopic GI biopsy specimens from 123 patients with GI symptoms after an allogeneic stem cell transplantation (allo-SCT; 2004-2017) were analyzed by IHC and PCR on 113 paraffin-embedded and 73 fresh samples. The results were then compared. Of the patients with macroscopic lesions in the mucosa and CMV-IHC-positive biopsy specimens (eg, “proven” CMV disease, n = 28), all but 1 were CMV-PCR positive. Of the patients without macroscopic lesions in the mucosa and CMV-IHC-positive biopsy specimens (eg, probable CMV disease, n = 4), only 1 was CMV-PCR positive. Eight patients had CMV-IHC-negative/CMV-PCR-positive gut biopsy specimens. These cases fall within the current definition of possible CMV disease. In 6 of these 8 cases (75%), the viral load in GI tissue was very high (>10,000 copies/µg). Taken together, the results from the proven and probable cases revealed that CMV-PCR shows the same sensitivity (100%), specificity (98%), and positive (93%) and negative predictive value (100%) as CMV-IHC. Detection of CMV in fresh GI mucosa by quantitative PCR is as useful as IHC for the diagnosis of GI CMV disease. The results show that quantitative PCR has the same sensitivity, specificity, and positive/negative predictive value as IHC.     

A Modified Intensive Strategy to Prevent Cytomegalovirus Disease in Seropositive Umbilical Cord Blood Transplantation Recipients

We previously demonstrated a lower rate of cytomegalovirus (CMV) reactivation and disease among seropositive umbilical cord blood transplantation (CBT) recipients receiving an intensive prophylaxis strategy consisting of ganciclovir on days ?8 to ?2 pretransplantation, high-dose valacyclovir post-transplantation, and twice-weekly serum CMV polymerase chain reaction testing. We hypothesized that a modified intensive strategy excluding pretransplantation ganciclovir would be similarly effective. We compared the risk of CMV reactivation, occurrence of CMV disease, and duration of anti-CMV therapy by day 100 post-CBT in patients receiving the modified intensive and intensive strategies. Forty patients received the modified intensive strategy, and 43 received the intensive strategy. There was no difference in the hazard for CMV reactivation (hazard ratio, 1.1; P?=?.77). No patients in the modified intensive cohort, but 2 patients in the intensive cohort, developed CMV disease (P?=?.53). There was no difference in the hazard for early (≤30 days post-CBT; P?=?.76) or high-level (>1000?IU/mL; P?=?.37) CMV reactivation. Patients in the modified intensive cohort had marginally higher CMV viral loads and percentage of days of CMV detection and treatment, although the contribution of pretransplantation ganciclovir to these differences is unclear. The overall percentage of treatment days was 32% in both cohorts after accounting for pretransplantation ganciclovir. In conclusion, exclusion of prophylactic ganciclovir before CBT did not impact the risk of CMV reactivation or disease, although CMV kinetics appeared to differ by prevention strategy. Best practices for CMV prevention will need further study as new prophylactic strategies become available.     

Glucocorticosteroids in Renal Transplantation

We have made an attempt to define a more ‘economical’ method for glucocorticosteroid (GS) administration in renal transplantation in man by shifting most of the GS to the immediate post-operative period. Thirty cadaver kidney recipients were prerandomized in 2 groups of 15 patients and monitored by transplant aspiration cytology (TAC). After transplantation the high-dose experimental group received 3.5 mg/kg/day of methyl-predinisolone (MP), tapered to 0.4 mg/kg/day in 14 days, and the low-dose control group 1.0 mg/kg/day, tapered to 0.4 mg/kg/day in 5 days. Upon rejection the patients in the high-dose experimental group were treated with at most 7 mg/kg/day of oral MP divided into four doses daily, whereas the low-dose control group received 1–3 intravenous boluses of 14 mg/kg (approximately 1000 mg/70 kg). Within 30 days after the transplantation, 7 clinically manifest rejection episodes and 2 episodes of in situ inflammation (without signs of rejection) were recorded in the experimental group, compared with 18 clinically manifest rejection episodes and 6 episodes of inflammation in the control group. The onset of the first episode of inflammation was delayed from 5.0±1.5 days in the control group to 6.6 ± 1.0 days (P=0.00) in the experimental group, and the onset of the first clinical rejection from 5.4±2.1 days to 8.5±1.0 days (P=0.00), respectively. TAC analysis documented that the first episode of inflammation was significantly smaller in the experimental group than in the control group (P=0.001), although the frequency of T lymphoblasts and monocytes was similar to that in the control group (P= 1.00 and 0.20), the frequency of in situ B lymphoblasts and lymphocytes was moderately depressed (P=0.04 and 0.04), and the accumulation of macrophages was both depressed (P=0.07) and delayed. After high initial MP administration the rejections were shorter and easier to overcome: the duration of the first inflammation episode was reduced from 10.5±4.4 days in the control group to 5.9±4.0 days (P=0.02) in the experimental group and the duration of clinical rejection from 8.4±4.1 days to 3.9±3.4 days, respectively (P=0.01). After 18-month follow-up study 12 patients in the experimental group had a life-supporting graft, compared with 7 patients in the control group. There was no difference in the frequency or type of complications between the two groups.     

African-Americans with End Stage Renal Disease in the Early Years of Kidney Transplantation

Introduction. The first successful kidney transplant in humans was performed in 1954. In the following 25 years, the biomedical, ethical, and social implications of kidney transplantation were widely discussed by both healthcare professionals and the public. Issues relating to race, however, were not commonly addressed, representing a “blind spot” regarding racial disparities in access and health outcomes. Methods. Through primary sources in the medical literature and lay press, this paper explores the racial dynamics of kidney transplantation in the 1950–1970s in the United States as the procedure grew from an experimental procedure to the standard of care for patients in end-stage renal disease (ESRD). Results & Discussion. An extensive search of the medical literature found very few papers about ESRD, dialysis, or renal transplant that mentioned the race of the patients before 1975. While the search did not reveal whether race was explicitly used in determining patient access to dialysis or transplant, the scant data that exist show that African-Americans disproportionately developed ESRD and were underrepresented in these early treatment populations. Transplant outcome data in the United States failed to include race demographics until the late 1970s. The Social Security Act of 1972 (PL 92-603) extended Medicare coverage to almost all Americans with ESRD and led to a rapid increase in both dialysis and kidney transplantation for African-Americans in ESRD, but disparities persist today.     

Glucocorticosteroids in Renal Transplantation

In a previous clinical trial we demonstrated that, by increasing the postoperative administration of methylprednisolone from 1.0 to 3.5 mg/kg/day, the onset of the first inflammatory rejection episode was significantly delayed and the size of the inflammation was reduced. The 'high initial' steroid treatment specifically depleted blast cells and macrophages from the in situ inflammatory infiltrate. In this trial we demonstrate that the 'high initial' glucocorticosteroid administration significantly improves 1-year cadaver allograft survival from 44% to 68% (P = 0.003) without increasing the number of complications. Although the 'high initial' steroid administration only partially overcomes the impact of HLA-AB incompatibility, it seems to overcome entirely the impact of absence of blood transfusions. The 'high initial' steroid administration also makes the first episodes of inflammation easier to overcome: less steroids are needed to counteract the first rejection, and, as a consequence, only 30% more steroids were used in the 'high initial' versus the 'low initial' steroid programme.