Human Cytomegalovirus Viral Load Research Articles

Molecular diagnosis of Cytomegalovirus infection: clinical performance of the Aptima transcription-mediated amplification assay toward conventional qPCR chemistry on whole blood samples.

In this paper, we describe the clinical performance of a novel transcription-mediated amplification (TMA) assay for the detection and quantification of human Cytomegalovirus (HCMV) DNA from whole blood samples. This is a pivotal analysis in immunocompromised patients [transplanted, HIV-positive, and Hematopoietic Stem Cell (HSC) recipients], and molecular tests with high sensitivity and specificity are necessary to evaluate the HCMV viral load in these patients. To our knowledge, this is the first in-depth evaluation of TMA chemistry for HCMV diagnosis on whole blood samples. Moreover, also technical aspects of this assay make it suitable for clinical diagnostics.

Prevalence of Human Cytomegalovirus Disease and its Related Factors in Renal and Bone Marrow Transplant Recipients in a Tertiary Hospital, Malaysia

Human cytomegalovirus (HCMV) infection may cause substantial morbidity and mortality after renal and bone marrow transplantation [1]. There are 3 major consequences of HCMV infection: HCMV disease with a wide range of clinical illnesses; superinfection with opportunistic pathogens; and injury to the transplanted organ [2]. Other than serological method to diagnose HCMV infection, viral load quantitation by real time polymerase chain reaction has been widely appreciated to diagnose and monitor the progress of viral infections. The aims of this study were to determine the incidence of HCMV infection in renal and bone marrow transplant recipients and to investigate its associations with HCMV disease, gender, and races.
 This retrospective cohort analysis involved 1520 blood samples from transplant recipients (renal, n = 164 and bone marrow, n = 182) from January 2020 to December 2021 collected from Virology Unit, Hospital Kuala Lumpur (NMRR ethical approval: NMRR-20-993-53201(IIR). The samples were analysed with quantitative polymerase chain reaction for HCMV DNA and the demographic, clinical and paraclinical aspects were evaluated. HCMV infection was present if the patient had positive HCMV viraemia and HCMV disease was diagnosed if HCMV infection was followed by clinical signs and symptoms. Statistical comparisons of patient demographics were performed with Chi-square tests for the categorical variables.
 The overall incidence of HCMV infection in the study group was 65% (225/346) where renal and bone marrow transplants account for 78.2% (176/225) and 21.7% (49/225) respectively. The incidence of HCMV infection in renal transplantation differed significantly by sex (p<0.05) where it was higher in males (71.8%) than in females (28.2%) but there was not statistically significant by sex in bone marrow transplantation in which males and females account for 61.2% and 38.7% respectively. The incidence of HCMV differed significantly (p<0.05) by races in both transplantation types as follows: 58% in Malay, 36% in Chinese, 5% in Indian and 1% in other indigenous races in renal transplantation while 59% in Malay, 29% in Chinese, 10% in Indian and 2% in other indigenous races in bone marrow transplantation. The incidence of HCMV disease differed significantly (p<0.05) by type of transplantation where it is higher in renal transplantation (30.9%) than in bone marrow transplantation (20.2%). The most seen symptoms were fever, generalised lethargy, and headache. Viral load of HCMV has been shown to be a major determinant factor in the severity and the manifestation of the HCMV infection[3].
 It is significantly higher in patients who develop HCMV disease[3]. Various risk factors have been described for the progress of symptomatic HCMV infection in organ transplant recipient[3]. The incidence of HCMV infection was higher in renal transplant as compared to bone marrow transplant among Malaysian. This study has shown that HCMV viral load has a significant association with age, gender and HCMV disease. Various syndromes can be caused by HCMV ranging from a mild fever to severe end-organ diseases. Treatment with anti-HCMV therapy results in decline in HCMV load, usually to undetectable.

The Viral Load of Human Cytomegalovirus Infection in Children following Hematopoietic Stem Cell Transplant by Chip Digital PCR.

Objective To detect viral load in human cytomegalovirus (HCMV) infection children after hematopoietic stem cell transplant (HSCT) by chip digital PCR (cdPCR). Methods The plasmid pUC57-UL83 containing the HCMV-UL83 gene and HCMV AD169 strain were used to evaluate the sensitivity of cdPCR. Either HSV-1, HSV-2, VZV, EBV, HHV-6, or HHV-7 was used to evaluate the specificity of HCMV cdPCR. The cdPCR was compared with quantitative PCR (qPCR) by detecting HCMV infection in 125 children's whole blood samples following HSCT. Results The limit of detection (LOD) of HCMV cdPCR was 103 copies/ml and the qPCR LOD was 297 copies/ml for plasmid pUC57-UL83. The result of HCMV cdPCR was 146 copies/ml for the HCMV AD169 strain, indicating that the sensitivity of cdPCR was higher than that of qPCR. There is no cross-reaction between HCMV cdPCR and other herpes viruses. The incidence of HCMV infection was 30.40% in 125 children following HSCT by cdPCR. The range of the HCMV viral load was from 107 copies/ml to 6600 copies/ml by cdPCR. Conclusions cdPCR is more sensitive than qPCR for detecting HCMV viral load. Furthermore, the cdPCR could be used to detect the viral load of HCMV infection before or after HSCT in children.

Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP.

Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3′-untranslated region (3′-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3′-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3′-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser536 residue and that p-Ser536 RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser536 through the downregulation of IE, and the binding of the resultant p-Ser536 RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p—together with p-Ser536 RelA/p65—can prevent lytic HCMV replication during acute and latent infection

Human cytomegalovirus (HCMV)-specific T cell but not neutralizing or IgG binding antibody responses to glycoprotein complexes gB, gHgLgO, and pUL128L correlate with protection against high HCMV viral load reactivation in solid-organ transplant recipients.

Immune correlates of protection against human cytomegalovirus (HCMV) infection are still debated. This study aimed to investigate which arm of the immune response plays a major role in protection against HCMV infection in kidney transplant recipients (n = 40) and heart transplant recipients (n = 12). Overall, patients were divided into 2 groups: one including 37 patients with low viral load (LVL), and the other including 15 patients with high viral load (HVL). All LVL patients resolved the infection spontaneously, whereas HVL patients were all treated with one or more courses of antivirals. In HVL patients, viral DNAemia, which was more than 100 times higher than LVL, appeared and peaked at significantly earlier times, but disappeared much later than in LVL patients. During a 1-year follow-up, all LVL patients had levels of HCMV-specific CD4+ (and CD8+ ) T cells significantly higher than HVL patients. On the contrary, titers of neutralizing antibodies and enzyme-linked immunosorbent assay-IgG antibodies to gB, gHgLgO, and pentamer gHgLpUL128L were overlapping in the 2 patient groups. In conclusion, while a valid HCMV-specific T-cell response was detected in more than 90% of LVL patients, >90% of HVL patients lacked an adequate T-cell response. Antibody responses did not appear to be associated directly or indirectly with protection.

Human cytomegalovirus glycoprotein polymorphisms and increasing viral load in AIDS patients

BackgroundMultiple strains infection of human cytomegalovirus (HCMV) was found to be correlated with increased viral load in immunodeficient patients. However, the pathogenic mechanism underlying this correlation remains unclear. To evaluate genetic polymorphisms of HCMV glycoprotein and their potential role in its viral load, HCMV glycoprotein B, N, and O (gB, gN and gO) genotypes was studied in the population of HCMV infected acquired immune deficiency syndrome (AIDS) patients. The association between glycoprotein polymorphisms and HCMV viral load was analyzed.MethodsThe genetic polymorphisms of glycoprotein from sera of 60 HCMV infected AIDS patients was investigated by multiplex nested PCR and sequencing. HCMV viral load was evaluated by quantitative PCR.ResultsgB1, gO1a, and gN4a were the predominant glycoprotein genotypes in HCMV infected AIDS patients and composed 86.96%, 78.8%, and 49.2%, respectively. Only gN4a genotype infection significantly increased viral load (P = 0.048). 71% (43/60) of HCMV infected AIDS patients were found to carry multiple HCMV strains infection. A novel potential linkage of gO1a/gN4a was identified from multiple HCMV infected patients. It was the most frequent occurrence, accounted for 51.5% in 33 patients with gO and gN genotypes infection. Furthermore, the gO1a/gN4a linkage was correlated to an increased viral load (P = 0.020).ConclusionThe gN4a correlates to higher level HCMV load in AIDS patients. Interestingly, a novel gO1a/gN4a linkage is identified from the patients with multiple HCMV strains infection and is also associated with an increased viral load. Therefore, the pathogenic mechanism underlying glycoprotein polymorphisms and interaction of variants should be analyzed further.

A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology

Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 106 International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

High Human Cytomegalovirus IgG Level is Associated with Increased Incidence of Diabetic Atherosclerosis in Type 2 Diabetes Mellitus Patients.

BackgroundAt present, whether human cytomegalovirus (HCMV) infection is associated with type 2 diabetes mellitus (T2DM) is debatable. The effect of active HCMV infection on glucose regulation has been poorly studied. Although HCMV infection is correlated with atherosclerosis in cardiovascular disease, the role of HCMV infection in the development of diabetic atherosclerosis in T2DM is unclear and is usually neglected by endocrinologists. The aim of this study was to assess the effects of HCMV infection on glucose regulation and the development of diabetic atherosclerosis in T2DM patients.Material/MethodsA total of 222 hospitalized T2DM patients were enrolled. Nested polymerase chain reactions were used to detect HCMV DNA extracted from peripheral blood leukocytes. Quantitative real-time PCR was used to determine viral load. HCMV IgG antibody concentrations were analyzed by chemiluminescence immunoassay.ResultsHCMV active infection, viral load, and HCMV IgG titers were not correlated with glucose regulation. Binary logistic regression demonstrated that the highest quartile of HCMV IgG concentration (>500 U/ml) was correlated with the incidence of diabetic atherosclerosis (OR: 8.0, 95%CI: 2.3–27.2), and that titer >127U/ml of HCMV IgG is an independent predictor for the development of diabetic atherosclerosis in T2DM patients (OR: 4.6, 95%CI: 1.9–11.3) after adjustment for all potential confounding factors.ConclusionsActive HCMV infection is unlikely to influence glucose regulation in T2DM. However, HCMV IgG titers are associated with the incidence of diabetic atherosclerosis, and titer >127U/ml of HCMV IgG might be an independent risk factor for the development of diabetic atherosclerosis in T2DM patients.

Human cytomegalovirus viral load in tumor and peripheral blood samples of patients with malignant gliomas

Malignant gliomas are the most common primary brain tumors in adults. The disease has no known etiology, progresses rapidly, and is fatal despite current therapies. Human cytomegalovirus (HCMV) is a beta herpes virus that is trophic for glial cells and infects 50% to 90% of the adult human population. HCMV-mediated disease in immunosuppressed patients has highlighted the possible role of this virus in the development of other diseases, particularly inflammatory diseases such as vascular diseases, autoimmune diseases, and certain malignancies. Sensitive detection of viral DNA, mRNA, and antigens in tumor tissues, as well as seroepidemiologic evidence, suggest a link between HCMV and several human malignancies. HCMV gene products are proposed to dysregulate multiple cellular pathways involved in oncogenesis, such as cell cycle regulation, apoptosis, migration, and angiogenesis. These theories, currently being researched, suggest that HCMV acts as an oncomodulator in malignancies. We investigated the association between HCMV infection and reactivation, and malignant gliomas. An open, matched case-control, parallel group pilot study was performed in a tertiary referral center. The HCMV viral load in peripheral blood and tumor samples of 19 patients newly diagnosed with glioblastoma multiforme was compared with a matched control cohort comprising 19 patients newly diagnosed with non-malignant brain tumors. There was no significant correlation between peripheral blood and tumor tissue HCMV viral load in patients with glioblastoma multiforme compared to the control cohort. The findings of the present study did not support an oncomodulatory role for HCMV in malignant gliomas.

Prevalence and risk factors for betaherpesvirus DNAemia in children >3 weeks and

Betaherpesviruses are established causes of morbidity and mortality in immunosuppressed patient groups but have been little studied in sub-Saharan Africa, the epicenter of the human immunodeficiency virus (HIV) pandemic. In this region, primary infections with human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) type 6 are endemic in infancy, but the clinical impact of these infections among pediatric inpatient groups is poorly characterized and assumptive, based largely on data from Western populations. We used TaqMan polymerase chain reaction to screen sera from a group of 303 pediatric inpatients aged between 3 weeks and 2 years, at the University Teaching Hospital in Lusaka, Zambia. We report the prevalence of DNAemia and viral loads within this patient group, and evaluate possible clinical associations/risk factors for betaherpesvirus infections in these hospitalized children. We detected betaherpesvirus DNAemia in 59.1% (179/303) of children. HCMV was the most prevalent (41.3%), followed by HHV-6B (20.5%), HHV-7 (20.1%), and HHV-6A (0.3%). HIV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.37-3.90; P = .002), being underweight (OR, 1.82; 95% CI, 1.06-3.12; P = .03), and an admission diagnosis of suspected meningitis (OR, 5.72; 95% CI, 1.07-30.5; P = .041) were independently associated with an increased odds of HCMV DNAemia. Conversely, HHV-6B and HHV-7 DNAemia were not associated with HIV, underweight, or admission diagnosis. Median HCMV viral load was moderately but significantly higher in HIV-infected children. Highly prevalent HCMV DNAemia was independently associated with HIV infection and being underweight across all age groups, and was also associated with meningitis, with previously underappreciated implications for the health and development of African children.

The increased sensitivity of human cytomegalovirus (HCMV) PCR quantitation in whole blood affects reproductive rate (Ro) measurement

In order to determine the effect of the increase in sensitivity of HCMV detection in whole blood compared to plasma on reproductive rate (Ro) measurement, an optimized human cytomegalovirus (HCMV) quantitative PCR assay was developed. The results presented in this study are summarized by the following three methodological improvements: (i) at values below the limit of quantitation (LOQ) of 60copies/ml, determination of HCMV load was more sensitive with whole blood than plasma, (ii) for the determination of viral load, whole blood was more sensitive than plasma below 1000copies/ml but little difference was observed above 1000copies/ml and (iii) the measurement of “Reproductive Rate” can be affected by imprecise measurement of HCMV viral load in either plasma or whole blood compartments depending on whether samples were taken from patients on antiviral treatment or from patients where HCMV load was rising. Taken together this study provides methodological improvements suggesting that below HCMV viral load levels of 1000copies/ml (1640IU/ml) both plasma and whole blood should be tested.

P.046 Serological micro-array for simultaneous detection of antibodies to measles, mumps and rubella viruses

Background: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. Objectives: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. Study design: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). Results: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24–48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. Conclusions: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.

Monitoring HCMV Infection With Quantitative Real-Time PCR in HCMV-Positive Orthotopic Liver Transplant Recipients, and Predictive Factors for Treatment of the First Episode of HCMV Viremia

We evaluated the relevance of human cytomegalovirus (HCMV) monitoring with quantitative real-time polymerase chain reaction in 42 consecutive HCMV positive liver transplant patients, and we analyzed the factors that determined the treatment of the first episode of HCMV DNAemia. No patients received anti-HCMV prophylaxis. HCMV infection monitoring was assessed every 2 weeks until day 90 and thereafter at every 3 to 4 weeks until day 180. HCMV infection was detected among 27 patients (64%, ie, 92/380 samples). Of these, 12 had their first HCMV DNAemia treated with IV gancyclovir (group I), whereas the other 15 patients were not treated (group II). Immunosuppressive treatment was not modified in cases of HCMV DNAemia. The median time between transplantation to the first CMV DNAemia was 37 days in group I and 52 days in group II (NS). Median HCMV viral load, whatever the treatment group and whatever the time of DNAemia, was 3 log copies/mL (0.48 to 5.80). Median HCMV viral load of the first positive DNAemia was 3.45 log copies/mL (1.69 to 5.80) in group I and 2.70 log copies/mL (1.15 to 3.94) in group II ( P = .01). Even though liver enzymes were increased in almost all patients presenting with HCMV infection, comparison of liver-enzyme levels and hematological parameters between the two groups at first HCMV viremia showed that alkaline phosphatase levels were significantly higher ( P = .0011) and hemoglobin levels were significantly lower in group I patients ( P = .0443). The only factor that predicted treatment for the first episode of HCMV DNAemia was an alkaline phosphatase level >150 UI/mL at the time of the first HCMV reactivation [odds ratio 20 (1.96 to 203.3); P = .01].

Breast milk as a source for acquisition of cytomegalovirus (HCMV) in a premature infant with sepsis syndrome: Detection by real-time PCR

Symptomatic disease due to human cytomegalovirus (HCMV) has been increasingly recognized in low birth weight (LBW) premature infants. Breast milk has been identified as a potential source for these infections. At approximately 60 days of life a LBW, premature infant was diagnosed with HCMV sepsis syndrome, prompting further investigation of the source for HCMV acquisition. The infant had received a combination of both fresh and frozen breast milk from his mother throughout his first 2 months of life. We utilized a real-time PCR assay for HCMV DNA to retrospectively examine viral genome copy number in serial samples of stored, frozen breast milk. This analysis indicated an increase in breast milk viral load over the first 45 days of life, heralding the HCMV sepsis syndrome that was observed clinically. Real-time PCR may be a useful tool in the evaluation of HCMV viral load in breast milk.

Follow-up of 28 HCMV seropositive renal-transplant recipients: comparison of clinical, biological and virological parameters in the groups of treated versus untreated infected patients

A pre-emptive strategy for prevention of Human Cytomegalovirus (HCMV) disease depends on accurate detection of HCMV infection and clinical and/or biological abnormalities. The aim of our study was to evaluate a therapeutic strategy based on the presence of either minimal clinical and/or biological symptoms or high HCMV viral load assessed by quantitative real-time PCR from whole blood as previously described. Between June 2002 and July 2003, 70 HCMV seropositive patients underwent a renal transplantation. Virological monitoring was performed every 2 weeks until day 90 then every 3-4 weeks until day 180. Biochemical and haematological parameters were also prospectively monitored. Twenty-eight patients (40%) showed at least one positive HCMV DNA in whole blood. Based on the following criteria: HCMV viral load greater than 4log(10)/ml, or the persistence of a HCMV DNAemia in two consecutive blood samples, or fever, or leucopenia or neutropenia, or increase in alanine aminotransferase level, 14 of the 28 patients received IV ganciclovir as a pre-emptive treatment. Immunosuppressive therapy and demographical data were comparable in both groups. As far as virological, haematological and biochemical parameters are concerned, no statistical significant difference was observed between treated and untreated patients. Moreover no adverse outcome was observed among untreated patients always having a HCMV viral load below 4log(10)/ml. This study reinforces the need of monitoring of HCMV seropositive renal-transplant recipients based on HCMV diagnosis and quantification by real-time PCR. The results showed that HCMV viraemic patients may benefit of absence of antiviral treatment when they have a low viral load (below 4log(10)/ml) and absence of clinical and/or biological abnormalities. Further studies are required now to validate the threshold value at which we should begin pre-emptive therapy.

HCMV DNA detection and quantitation in the plasma and PBL of lung transplant recipients: COBAS Amplicor HCMV monitor test versus in-house quantitative HCMV PCR.

Human cytomegalovirus (HCMV) reactivation may cause severe disease in immunosuppressed patients. Quantitation of HCMV viral load in the blood has been shown to be important in predicting for HCMV disease, however the particular blood compartment that should be tested, plasma versus peripheral blood leukocytes (PBL), has been subject to debate. To simultaneously compare HCMV viral loads in the PBL using an in-house quantitative HCMV polymerase chain reaction (PCR) assay and in the plasma using the commercially available COBAS Amplicor HCMV monitor test, in a cohort of lung transplant recipients (LTR). Sequential paired plasma and PBL samples were collected (n=98) from a total of 21 LTR during the first 6 months post lung transplantation. HCMV viral loads were assessed in the PBL using an in-house quantitative HCMV PCR assay and the plasma using the COBAS Amplicor HCMV monitor test. HCMV disease in LTR was defined as histopathologically proven HCMV pneumonitis. HCMV deoxyribonucleic acid (DNA) was detected in 39/98 (40%) of total samples with excellent agreement between the two strategies. HCMV was detected in both the plasma and PBL in 38/39 (97%) of HCMV positive samples. HCMV viral loads were higher in patients with HCMV pneumonitis in both the PBL and plasma compared to patients without HCMV pneumonitis. Greater than 10-fold increases in HCMV DNA levels had a sensitivity of 88% and a specificity of 73% in the plasma for HCMV pneumonitis and a positive and negative predictive value of 70 and 89%, respectively. Greater that 10-fold increases in HCMV DNA levels in the PBL had a sensitivity of 88% and a specificity of 64%, for HCMV pneumonitis and a positive and negative predictive value of 64 and 88%, respectively. Acknowledging the difference in assay methods used for HCMV DNA quantitation, the in-house PCR PBL assay and the COBAS Amplicor HCMV monitor plasma assay predicted equally well for HCMV pneumonitis in LTR.

HCMV DNA detection and quantitation in the plasma and PBL of lung transplant recipients: COBAS Amplicor HCMV monitor test versus in-house quantitative HCMV PCR

Background: Human cytomegalovirus (HCMV) reactivation may cause severe disease in immunosuppressed patients. Quantitation of HCMV viral load in the blood has been shown to be important in predicting for HCMV disease, however the particular blood compartment that should be tested, plasma versus peripheral blood leukocytes (PBL), has been subject to debate. Objectives: To simultaneously compare HCMV viral loads in the PBL using an in-house quantitative HCMV polymerase chain reaction (PCR) assay and in the plasma using the commercially available COBAS Amplicor HCMV monitor test, in a cohort of lung transplant recipients (LTR). Study design: Sequential paired plasma and PBL samples were collected (n=98) from a total of 21 LTR during the first 6 months post lung transplantation. HCMV viral loads were assessed in the PBL using an in-house quantitative HCMV PCR assay and the plasma using the COBAS Amplicor HCMV monitor test. HCMV disease in LTR was defined as histopathologically proven HCMV pneumonitis. Results: HCMV deoxyribonucleic acid (DNA) was detected in 39/98 (40%) of total samples with excellent agreement between the two strategies. HCMV was detected in both the plasma and PBL in 38/39 (97%) of HCMV positive samples. HCMV viral loads were higher in patients with HCMV pneumonitis in both the PBL and plasma compared to patients without HCMV pneumonitis. Greater than 10-fold increases in HCMV DNA levels had a sensitivity of 88% and a specificity of 73% in the plasma for HCMV pneumonitis and a positive and negative predictive value of 70 and 89%, respectively. Greater that 10-fold increases in HCMV DNA levels in the PBL had a sensitivity of 88% and a specificity of 64%, for HCMV pneumonitis and a positive and negative predictive value of 64 and 88%, respectively. Conclusions: Acknowledging the difference in assay methods used for HCMV DNA quantitation, the in-house PCR PBL assay and the COBAS Amplicor HCMV monitor plasma assay predicted equally well for HCMV pneumonitis in LTR.

Development of a real-time PCR procedure including an internal control for the measurement of HCMV viral load

Human cytomegalovirus (HCMV) infections are frequent in immuno-compromised patients. The recent development of real-time PCR procedures that allow the rapid quantification of genome load will be helpful for accurate monitoring of these infections. Two extraction procedures were evaluated using 30 blood samples that were processed pure and diluted (1/10). Repeatability and reproducibility of the quantitative PCR procedure using an internal control for amplification were analysed, and its sensitivity compared to a qualitative PCR procedure using 50 HCMV culture positive blood samples. The real-time PCR and qualitative PCR procedures were positive in 46 and 48 of the samples tested, respectively. Discrepancies were observed for samples with a low viral load. The sensitivity of the real-time PCR procedure was evaluated at 500 HCMV DNA copies per ml of sera. The use of an internal control concomitantly processed during the HCMV quantification did not alter the sensitivity of the procedure, and was relevant for the detection of putative PCR inhibitors that may interfere with the amplification process. This procedure was used to measure genome load in two bone marrow transplant patients with HCMV disease, confirming that this new PCR procedure should be used widely for diagnosing and monitoring HCMV infections in transplant patients.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay.

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 10(6) leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 10(6) leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Human cytomegalovirus glycoprotein B genotypes in blood of AIDS patients: Lack of association with either the viral DNA load in leukocytes or presence of retinitis

It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty-four blood samples were obtained from 54 HIV- and HCMV-infected patients. Twenty-seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR-amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative-PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR-amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV-infected subjects at risk of HCMV disease.