Detection Of Human Cytomegalovirus Infection Research Articles

CD64 expression on polymorphonuclear cells as a potential marker for monitoring the human cytomegalovirus replication after kidney transplantation

Human cytomegalovirus (HCMV) infection is the main cause of morbidity in kidney transplant recipients. This study aims to investigate if CD64 expression on polymorphonuclear (PMN) cells is useful for the detection of HCMV infection in eleven kidney recipients during sixty days. From the total patients, nine were positive for both pp65 antigenemia and HCMV by quantitative polymerase chain reaction (qPCR), all of which had circulating neutrophils expressing CD64 3-4 weeks prior to pp65 antigenemia peak. These results suggest that quantification of PMN CD64 together with pp65 antigenemia could be useful for the early diagnosis of HCMV after transplantation.

Rapid detection of active human cytomegalovirus infection in pregnancy using loop-mediated isothermal amplification

Understanding the association between congenital human cytomegalovirus (HCMV) infection and active maternal HCMV infection during pregnancy is important for maternal and neonatal healthcare. In the present study, a loop-mediated isothermal amplification (LAMP) method was established for the detection of CMV DNA from whole blood or amniotic fluid samples, using reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the CMV LAMP assay detection was specific for CMV DNA, whereas it did not detect viral DNA from herpes simplex type 1 (HSV-1), HSV-2, varicella zoster virus, HSV-6 or HSV-7. Sensitivity determination using serially-diluted CMV glycoprotein B-containing plasmids, demonstrated that >10 copies per tube were detectable using the CMV LAMP method. Furthermore, the detection results, using the LAMP method for 336 whole blood samples, demonstrated that at a threshold of 10(1)-10(4) copies per tube, the sensitivity of this method was 86.96-100%, the specificity was 97.24-100%, the positive predictive value was 76.92-100% and the negative predictive value was 99.05-100%. The results for 11 amniotic fluid samples from pregnant women with whole blood CMV-positive and 15 control amniotic fluid samples, indicated that the CMV LAMP assay was sensitive and specific for CMV detection. In conclusion, in the present study, a CMV LAMP method was developed, which was shown to be sensitive, specific and efficient in the detection of HCMV infection. Furthermore, CMV LAMP is capable of detecting active CMV infection in pregnant women. Therefore, the current study provides novel insights into diagnostic approaches for active CMV infection in pregnant women.

Monitoring of Human Cytomegalovirus Infection in Bone Marrow and Liver Transplant Recipients by Antigenaemia Assay and Enzyme-Linked Immunosorbent Assay

Human cytomegalovirus (HCMV) infection is a common complication in transplant recipients. Sensitive, specific and timely diagnostic tests for the detection of HCMV infection remain essential for successful therapy. The results of three tests to detect HCMV in bone marrow and liver transplant recipients were compared: a pp65 antigenaemia assay, an immediate-early (IE) antigenaemia assay and an anti-HCMV immunoglobulin M (IgM) antibody enzyme-linked immunosorbent assay (ELISA). Of 1344 samples, 911 (67.8%) and 917 (68.2%) samples were positive for pp65 and IE, respectively. The coincidence level was 85.1%. There was no statistical difference after transplantation to the first positive detection of HCMV (mean first checkout time) between the pp65 and IE antigenaemia assays. Moreover, the levels of HCMV detected by the pp65 and IE antigenaemia assays were significantly correlated. The HCMV-positivity rate as detected by the anti-HCMV IgM ELISA was 11.1%, which was significantly different from the IE and pp65 antigenaemia assays. We suggest that the IE antigenaemia assay could replace the pp65 antigenaemia assay for monitoring active HCMV infection and early detection of HCMV infection.

New cell-based indicator assays for the detection of human cytomegalovirus infection and screening of inhibitors of viral immediate-early 2 protein activity

Expression of early (E) genes of human cytomegalovirus (HCMV) is stimulated cooperatively by the activities of host cell transcription factors and the viral immediate-early 2 (IE2) protein. Taking advantage of the IE2-dependent inducibility of E gene promoters, in this study, we generated cell-based assays in which the expression of the enhanced green fluorescence protein (EGFP) reporter gene was driven by the UL54 or UL112/113 E promoters. Cell clones derived from a stably transfected human cell line permissive to HCMV replication showed a specific and inducible dose- and time-dependent EGFP response to HCMV infection. The sensitivity of these indicator cells for detecting infectious particles of clinical isolates of HCMV was comparable to that of a conventional plaque assay. The HCMV-induced EGFP expression was completely prevented by treatment of indicator cells with fomivirsen, an antisense oligodeoxynucleotide designed to block IE2 expression, and this inhibitory activity was also observed when the IE2 protein alone was constitutively expressed in EGFP indicator cells. The EGFP-based cell assays have proved to be a rapid, sensitive, quantitative and specific system for detection of HCMV and selection of antivirals. These new cell-based assays can be exploited as functional assays to detect infectious HCMV particles, as well as to screen antiviral compounds that interfere with IE2 activity.

Early detection of human cytomegalovirus infection after kidney transplantation by nucleic acid sequence-based amplification.

The early detection of human cytomegalovirus infection after organ transplantation is a prerequisite for effective antiviral therapy. We evaluated the diagnostic value of monitoring the viral immediate-early (IE) 1 mRNA expression in blood leukocytes by nucleic acid sequence-based amplification (NASBA). Nucleic acids were isolated from 489 blood samples collected from 42 kidney transplant recipients and subjected to amplification by IE NASBA. The IE NASBA results were compared to those from pp67 NASBA, pp65 antigenemia, cell culture (DEAFF and CPE), and serology. IE NASBA proved to be the most sensitive assay which detected the onset of both primary and secondary cytomegalovirus infection significantly earlier than the other assays. The early detection of cytomegalovirus infection with IE NASBA would enable the start of effective antiviral therapy at an early state of infection to prevent cytomegalovirus disease in patients at risk.

Evaluation of a microtiter plate fluorescent-antibody assay for rapid detection of human cytomegalovirus infection.

The use of monoclonal antibody (MAb) p63-27, which is reactive with the major immediate-early human cytomegalovirus (HCMV) protein pp72, was explored for the rapid diagnosis of HCMV viruria. The rapid assay detected all but 1 of 19 specimens identified by standard virus isolation methods from 1,676 newborn urine specimens, achieving a sensitivity of 94.5% and a specificity of 100%. The monoclonal antibody recognized 260 randomly obtained clinical isolates of HCMV, indicating the suitability of this reagent for use in screening assays. The sensitivity of the microtiter plate method declined rapidly for specimen from older infants and children with congenital CMV infection and virus-infected children attending a day-care center and was judged to be unacceptable for screening populations in this age group.

Detection of human cytomegalovirus infection in paraffin sections by modified Steiner silver stain for LM and TEM

Human cytomegalovirus (HCMV) infection is encountered more frequently in surgical specimens due to the increased number of immunosuppressed patients (organ transplants or HIV positive). Previous studies showed that HCMV infected cells in paraffin sections can be precisely labelled by in situ hybridization or by Grocott's methenamine silver stain for LM and subsequently confirmed by TEM. However, further studies indicate HCMV infection can also be identified by modified Steiner silver stain3 but in a different manner.Modified Steiner silver stained paraffin sections can be processed for TEM as follows: Coverslips are removed by immersion in xylene. A few drops of 1% OsO4, freshly dissolved in xylene, is then pipetted onto each slide for 5 minutes. Aqueous OsO4 can not be used as it will destain the sections instantly. Slides are rinsed in xylene, xylene/acetone and then 100% acetone. The sections are infiltrated with epoxy resin/acetone, embedded in epoxy resin, polymerized and subsequently separated from slides by heat as previously described.

Detection of human cytomegalovirus infection in paraffin sections by Grocott’s methenamine silver stain for light and electron microscopy

Grocott's methenamine silver stain (GMS) has been used histologically for identifying Pneumocystis carinii and fungal infections in paraffin sections of surgical specimens. However, current studies indicate that aggregates of human cytomegalovirus (HCMV) in the cytoplasm of infected cells are also stained by GMS and can be detected by LM. Since precipitated silver particles are electron dense, the presence of silver labelled HCMV virions can be easily located and confirmed by TEM.GMS and light green stained paraffin sections positive for HCMV can be processed for TEM either by conventional procedures or as follows: Coverslips are removed by immersion in xylene. A few drops of 1% 0s04, freshly dissolved in xylene, are then pipetted onto each slide for 5 minutes. Prolonged osmication is to be avoided as it will cause reduction of the silver deposits. Slides are then rinsed in xylene and changed to 100% acetone. Substitution of acetone for xylene prior to resin infiltration will produce a better quality block for sectioning. The sections are embedded, polymerized and subsequently separated from slides as previously described. The light green staining is retained in the epoxy block, which facilitates tissue orientation and selection of areas for thin-sectioning.

Comparison of in situ DNA hybridization and immunological staining with conventional virus isolation for the detection of human cytomegalovirus infection in cell cultures

The sensitivity of immunochemical staining and in situ DNA hybridization for the detection of human cytomegalovirus (HCMV) was compared with that of virus isolation. Human diploid fibroblasts were infected with serial, 10-fold dilutions of HCMV strain AD169 and examined at various intervals between 1 and 42 days after inoculation, using the three methods being compared. HCMV-DNA was detected by in situ hybridization using a biotin-labeled HCMV probe and CMV early antigen (EA) by immunochemistry using a specific monoclonal antibody. During the first 2 days after inoculation detection of EA appeared to be the most sensitive method. After the fifth day the sensitivity of the immunochemical and in situ hybridization methods was similar and equalled that of conventional virus isolation. However, 2–5 times more HCMV-DNA than HCMV-EA positive cells were detected. Our results indicate that both detection of HCMV-EA by immunological staining and HCMV-DNA by in situ hybridization are suitable methods for rapid and sensitive detection of HCMV infections.