Polymerase Chain Reaction Assays for the Detection of Cytomegalovirus in Organ and Bone Marrow Transplant Recipients

Cytomegalovirus (CMV) infection is ubiquitous and results in a wide spectrum of clinical manifestations ranging from asymptomatic infection to severe life threatening disease. Infection in normal children and adults usually causes no symptoms but in the immuncompromised host, CMV may result in severe opportunistic infections with high morbidity and mortality. Historically, virus detection was dependent on culture of the virus or on a centrifugation culture system referred to as a shell vial assay. The shell vial assay frequently lacked sensitivity and was unable to detect infection in its early phase. Also, as with culture assays, the results were affected by antiviral therapy. The CMV antigenemia assay was developed to provide more rapid results and has gained wide usage. This assay is limited to detection of the virus in white blood cells and is more sensitive than culture or the shell vial assay.

Application of the polymerase chain reaction (PCR) to these problems has resulted in the development of assays for CMV which are more sensitive than previously available methods. This method employs liquid hybridization with ³²P labeled probes and gel retardation analysis for detection of amplified DNA specific for each virus.

A comparison of the detection of CMV by an antigenemia assay or the PCR method in the leukocytes of renal transplant patients revealed that the PCR assay detects cytomegalovirus earlier and more consistently than the antigenemia assay.

Finally, the application of a fluorescent dye detection system and image analysis of the acrylamide gel with a laser scanner provides additional sensitivity to the detection of cytomegalovirus, as well as avoiding the use of radioactivity, making the assay more adaptable to the clinical laboratory.     

Cytomegalovirus in Bone Marrow Cells Correlates with Cytomegalovirus in Peripheral Blood Leukocytes

In allogeneic hematopoietic stem cell transplant recipients with bone marrow (BM) suppression, cytomegalovirus (CMV) pp65 antigenemia and DNA were detectable in peripheral blood leukocytes (PBL) and BM cells. A relationship between CMV infection of PBL and BM cells has been found.     

Co-Infections with Cytomegalovirus and Human Herpesvirus Type 7 in Adult Polish Allogeneic Haematopoietic Stem Cell Transplant Recipients

Human herpesvirus 7 (HHV-7) is widespread around the world and may also be a possible cofactor for cytomegalovirus (CMV) infection in haematopoietic stem cell transplant (HSCT) recipients. In case of viral diseases where specific treatment is available, real-time PCR assays constitute reliable diagnostic tools enabling timely initiation of appropriate therapy and rapid assessment of the efficacy of antiviral treatment strategies. The presence of CMV and HHV-7 was confirmed by the detection of viral DNA isolated from 1,027 plasma samples. A group of 69 allogeneic HSCT (alloHSCT) recipients was examined in early post-transplant period using quantitative real-time PCR methods. Within the study period, 62 % of patients had at least once CMV DNA-emia, while HHV-7 DNA was found in 43 % of subjects. Co-infection between these β-herpesviruses was detected in the plasma samples collected from 18 patients (26 %). Patients with concomitant HHV-7 DNA-emia had significantly higher number of CMV DNA copies compared with those without HHV-7 infection (1986 vs. 432 copies/ml, p < 0.001) but there was no difference in duration of CMV DNA-emia between these groups. On the other hand, while the load of HHV-7 DNA was comparable between patients with CMV DNA-emia and without CMV DNA-emia, the duration of HHV-7 DNA-emia was significantly longer in the first group (38.5 vs. 14 days, p < 0.001). HHV-7 DNA-emia is very frequently detected in Polish alloHSCT recipients. In those, who have subsequent CMV reactivation, the coexistence of the viruses may negatively affect the kinetics of infection with either of them. Therefore the investigation of concomitant HHV-7 DNA-emia could affect the prognosis of post-transplant patients suffering from CMV reactivation.     

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.     

Detection of Cytomegalovirus Disease by Real-Time Quantitative PCR Targeting Immediate Early Gene (ppUL83) in Different Samples among Post-Renal-Transplant Recipients

Renal transplantation is a treatment option for end-stage renal disease (ESRD). Cytomegalovirus (CMV) infection was analysed among symptomatic and asymptomatic post-renal-transplant recipients (PRTRs). A total of 30 PRTRs were enrolled. DNA was extracted and quantitative real-time PCR for CMV (CMV R-Gene, France) targeting ppUL83 gene was performed on whole blood, urine and saliva. The detection rate of CMV was found to be 27% (n = 8) in different samples, including whole blood, urine and saliva. Among 30 PRTRs, 53% (n = 16) of the PRTRs did not shed virus in saliva. About 7% of CMV was detected only in saliva among PRTRs who were symptomatic.     

Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis.     

肾移植术后受者人巨细胞病毒感染与其它机会感染的研究

应用聚合酶链反应（ＰＣＲ）技术检测６５例肾移植受者（ＲＴＲ）尿中人巨细胞病毒（ＨＣＭＶ）－ＤＮＡ，配合捕获ＥＬＩＳＡ法及间接ＥＬＩＳＡ法检测ＨＣＭＶ－ＩｇＭ及ＩｇＧ，诊断ＨＣＭＶ感染，感染率为６０％（３９／６５）。应用临床及实验室方法诊断其它病原体（如革兰氏细菌，结核杆菌，霉菌，ＨＳＶ及ＶＺＶ等）所致的机会感染，结果表明，ＨＣＭＶ感染组机会感染率明显高于ＨＣＭＶ未感染组（Ｐ＜０．０１）。对其机理的分析表明，ＨＣＭＶ感染后外周血Ｔ细胞亚群产生了变化，即ＨＣＭＶ感染组ＣＤ４水平显著低于ＨＣＭＶ未感染组，ＣＤ８水平显著增高，导致ＣＤ４／ＣＤ８值显著降低（Ｐ均小于０．０１），说明ＨＣＭＶ感染组患者细胞免疫受到严重抑制，招致其它病原体的机会感染。     

Long-Term Persistence of Immunoglobulin A (IgA) and IgM Antibodies against Human Cytomegalovirus in Solid-Organ Transplant Recipients

The retrospective analysis of 494 solid-organ transplant recipients revealed that during the follow-up period (mean duration, 3.2 years) 184 (88%) of 209 anti-human cytomegalovirus (HCMV) immunoglobulin A (IgA)-positive patients remained IgA positive, as did 128 (74.85%) of 171 anti-HCMV IgM-positive patients. We conclude that anti-HCMV IgA and IgM testing for management of clinically relevant HCMV infections in solid-organ transplant recipients is dispensable.     

Automated Nucleotide Sequencing Reveals Substantial Disparity Between the HLA-A2 Genes of Bone Marrow Transplant Recipients and Donors

ABSTRACT: HLA disparity is associated with immunological complications after bone marrow transplant and it has been demonstrated that a single amino acid substitution can dramatically alter the function or allorecognition of an HLA molecule. Current serological methods for typing Class I HLA do not distinguish between most HLA-A2 variants which can differ by 1–8 amino acid residues. HLA-A2 disparity between bone marrow transplant patients and donors was investigated using automated nucleotide sequencing of the entire coding region of HLA-A2 genes. A total of 122 HLA-A2 alleles were sequenced from 47 patient-donor pairs (94 individuals). HLA-A2 disparity was observed in 10 of 47 pairs (21.3%) and consisted of HLA-A*0201 mismatched with 0202 (n = 2), 0205 (n = 3), 0206 (n = 3), 0217 (n = 1) or 0221 (n = 1). Four of 6 (66.7%) non-Caucasian or mixed race pairs were HLA-A2 disparate, while 6 of 36 (16.7%) Caucasian pairs were HLA-A2 disparate (p = 0.008). Among all individuals HLA-A*0201 was the most frequently observed allele (90.0%) while 0202 (1.6%), 0205 (2.5%), 0206 (4.1%), 0217 (0.8%) and 0221 (0.8%) were also observed. This study illustrates the diversity of HLA-A2 in non-Caucasian individuals and suggests that HLA-A2 subtyping for applications such as bone marrow transplantation, especially in non-Caucasian or mixed-race donor-recipient pairs, may be important.     

Quantification of Human Cytomegalovirus DNA in Amniotic Fluid of Mothers of Congenitally Infected Fetuses

A quantitative PCR assay was used to quantitate human cytomegalovirus DNA in amniotic fluid of mothers of 21 fetuses with congenital infection. Seven fetuses presented ultrasound abnormalities or were born with symptoms, whereas 14 fetuses were subclinically infected. Although the median DNA level was higher in symptomatic fetuses, the difference was not statistically significant (P = 0.09).     

Puppet Therapy with Pediatric Bone Marrow Transplant Patients

The authors' experience in developing and implementing a formof short-term puppet therapy for children undergoing bone marrowtransplantation is reviewed.     

Comparative Quantitation of Cytomegalovirus (CMV) DNA in Solid Organ Transplant Recipients with CMV Infection by Using Two High-Throughput Automated Systems

Cytomegalovirus (CMV) DNA quantitation in clinical specimens is progressively becoming a cornerstone in the diagnosis and management of CMV infection in the immunocompromised host. We evaluated two automated and reproducible PCR tests, the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and the COBAS AMPLICOR CMV Monitor (Roche Diagnostics, Pleasanton, Calif.), for the detection of CMV DNA in blood samples from transplant recipients with CMV infection as determined by shell vial culture. Following a log transformation analysis, the mean CMV DNA in plasma (PL), whole blood (WB), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) using the LightCycler was 6.79 copies per ml, 7.23 copies per ml, 6.38 copies per 2 x 10(6) cells, and 6.27 copies per 2 x 10(6) cells, respectively. This compares to 7.86 copies per ml, 8.37 copies per ml, 7.59 copies per 2 x 10(6) cells, and 7.44 copies per 2 x 10(6) cells, respectively, using COBAS AMPLICOR CMV Monitor. While higher CMV DNA levels were observed for the various blood compartments analyzed using COBAS AMPLICOR CMV Monitor, a high degree of correlation was evident between the two automated systems (jackknife correlation r = PL 0.77 [95% confidence interval (CI); 0.64, 0.90], WB 0.77 [95% CI; 0.62, 0.92], PBL 0.77 [95% CI; 0.67, 0.88], and PBMC 0.81 [95% CI; 0.72, 0.89], all P < 0.001). Therefore, we conclude that either automated diagnostic system is accurate for CMV DNA quantitation.     

Performance of an In-House Real-Time PCR Assay for Detecting Cytomegalovirus Infection among Transplant Patients from a Tertiary Care Centre

Background: Quantitative Cytomegalovirus (CMV) polymerase chain reactions are increasingly being used for monitoring CMV DNAemia in haematopoietic stem cell transplants and solid organ transplants. Objective: In this study, a commercial CMV viral load assay was compared with an in-house viral load assay. Materials and Methods: A total of 176 whole-blood samples were tested for CMV DNAemia using both assays. Results: Our evaluation showed a difference of 1 log₁₀ copies/ml between the two assay systems in determining CMV viral loads in the clinical samples. Conclusion: The in-house viral load assay had a better correlation with clinical findings compared to the commercial assay. Quality assessment of these assays was done by the United Kingdom National External Quality Assessment Scheme (UKNEQAS), an external proficiency testing programme, and by the National Institute for Biological Standard and Control (NIBSC) standard. For UKNEQAS and NIBSC standards, the bias between the assays was 0.73 log₁₀ and 0.85 log₁₀, respectively. This difference is well within the acceptable range already reported in the literature.     

Utility of Major Leukocyte Subpopulations for Monitoring Secondary Cytomegalovirus Infections in Renal-Allograft Recipients by PCR

The feasibility of the major peripheral blood leukocyte (PBL) subsets for use in qualitative and quantitative PCR to monitor secondary cytomegalovirus (CMV) infection and ganciclovir therapy was assessed with 188 blood samples derived from 40 CMV immunoglobulin G-positive renal-allograft recipients. In pp65 antigen-positive patients all leukocyte fractions, but only 79.5% of plasma preparations, were PCR positive. In pp65 antigen-negative samples from patients after antiviral treatment only 7.3% of polymorphonuclear cell (PMNL) samples, but 81.8% of peripheral blood mononuclear cells (PBMC), and 10.9% of plasma samples remained PCR positive. Similarly, in patients with latent infections only 5.0% of PMNL, but 51.7% of PBMC preparations, and 8.0% of plasma samples were PCR positive. Regarding patients with active CMV infection, CMV DNA copy numbers in PMNL correlated significantly with pp65 antigen-positive cell counts before and after onset of ganciclovir therapy. Significant differences in CMV DNA copy numbers in PMNL and plasma were observed (i) between patients with symptomatic infection and those with asymptomatic infection and (ii) between patients with active infection and those with latent infection. In contrast, PBMC harbored equally low CMV DNA levels both in patients with active infection and those with latent infections, and no decline of CMV DNA load in PBMC was observed during antiviral treatment. We conclude that detection of CMV DNA in PMNL, not in PBMC, is associated with active infections and is more sensitive than detection of CMV DNA in plasma. Negative PCR results for PMNL after antiviral therapy indicate recovery, and fewer unwanted positive results occur compared to PBMC and plasma. Therefore, purified PMNL should be preferred for analysis by qualitative CMV PCR to avoid unwanted positive results. The CMV DNA load in PBMC compared with that in PMNL is negligible during active infection, so mixed PBL are sufficient for use in quantitative PCR.