Determination of human cytomegalovirus pp65 antigenemia among renal transplant patients

Introduction

More than 80% of the adult population worldwide is infected with human cytomegalovirus (HCMV). Primary infections occur early in the childhood and may cause exanthema subitum. HCMV infection is an important cause of morbidity and mortality in immunosuppressed organ transplant recipients.[1–3]

As the diagnosis of HCMV infections cannot be made reliably on clinical grounds alone, laboratory confirmation is required. The most specific laboratory method for diagnosis of HCMV infection is isolation of the virus in culture. Isolation of HCMV from blood leukocytes (CMV viremia) is considered the most reliable marker of disseminated CMV infection, and predicts invasive CMV disease.[4–8] HCMV can infect a number of cell types, namely, fibroblasts, epithelial and endothelial cells, and smooth muscle cells.[9] In particular, HCMV has been shown to infect and replicate in endothelial cells of the vascular tree. HCMV is also present in peripheral myeloid cells of 50-90% of normal individuals. In disseminated infection, cytomegalic endothelial cells may circulate in the peripheral blood, and virus dissemination is mediated by peripheral blood leukocytes (PBLs) carrying infectious virus acquired from infected endothelium and transmitting the infection to uninfected endothelial cells.[10–12] Isolation of HCMV from human PBLs has been documented. Successful isolation was reported for various leukocyte subpopulations such as granulocytes, T lymphocytes, or monocytes.[1314] pp65 antigen of HCMV is recognized by more than 70% of HCMV-specific circulating T lymphocytes (CTLs). As pp65 is processed and presented before endogenous viral replication, pp65-specific CTLs may initiate the HCMV spread.[15–20]

The detection of HCMV pp65 antigenemia is widely used for monitoring CMV infection and guiding preemptive therapy in patients at risk of developing HCMV disease, as a semiquantitative analysis of HCMV viral replication.[2122] The CMV pp65 antigenemia assay, which quantitates the number of HCMV-infected leukocytes in peripheral blood, has proven efficacy in the detection and monitoring of this virus infection in immunocompromised patients.[23–26] This study was undertaken to determine the significant pp65 antigenemia count among renal transplant patients, by evaluating the test against viral isolation.

Materials and Methods

Results

Comparison of pp65 antigenemia assay and viral isolation

Among the 49 peripheral blood specimens used for inoculation, the cultures showed megaloblastic changes in 21 (42.86%) specimens [Figure 1]. Patients under group A had a cell count ranging from 2 to 10 cells, with a mean 6.3. This group of patients had an isolation rate of 20%. Group B had a cell count ranging from 10 to 25 cells, with a mean of 15.2, and there were 7 (28%) isolates from this group of patients. Group C had a count ranging from 25 to 50 cells with a mean cell count of 39.3, and 4 (66.67%) specimens of the virus were isolated. Group D had a cell count >50 cells with a mean count of 229.11, and cultures were pisitive in 8 (88.89%) specimens [Table 1]. Isolation of the virus was further confirmed on RT-PCR assay performed on the cell culture isolates.

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Figure 1

(a) Uninfected cell control. (b) Infected fibroblast cultures

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Table 1 Results of patient groups based on pp65 antigenemia assay and culture isolation

Inactivation of the virus was ruled out as all the isolates were stored at -80°C until processed. Each lot of fibroblast cell culture without exposure to leukocytes was harvested and DNA extracted from them was subjected to rule out the presence of endogenous HCMV infection.

Discussion

Gregory et al.,[28] compared three different assays designed for the rapid and sensitive detection of CMV in blood: quantitative shell vial culture (QSVC), PCR, and the pp65 antigenemia assay. It was found that PCR was the most sensitive of the three, allowing recognition of CMV in blood specimens from a larger number of patients and at an earlier time after transplantation than the other assays, and PCR also remained positive for the longest time after transplantation. Quantitative shell vial culture and the antigenemia assay were comparable both in the number of patients in whom CMV was detected in blood and in the time to detection. The advantage of culture was highlighted by the observation that when antiviral therapy was used, QSVC tended to become negative first, while PCR stayed positive the longest. The study finally concluded that the pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.[28]

Reina et al.[29] concluded that the inoculation of all of the leukocytes extracted from blood samples in the SVC seems to produce a slight increase in the sensitivity of the cell culture and that the SVC becomes positive before the antigenemia for the detection of CMV in peripheral blood, especially in the first blood sample.[29]

In another study, assays that analyzed 10,000 leukocytes had moderate analytical sensitivity, thereby exhibiting moderate positive predictive value (PPV) and specificity for the prediction of CMV disease in patients after stem cell transplantation. The sensitivity of RT-PCR depends on the assay conditions. PCR tests with high sensitivity have a low PPV and specificity, and threshold values need to be evaluated under clinically relevant conditions to obtain an appropriate specificity and PPV as close to 100% as possible to avoid unnecessary CMV treatment. In addition to the question of sensitivity, the availability of patient specimens may influence the choice of a test. pp65 antigen detection requires leukocytes from freshly collected blood samples, which cannot be stored or frozen. In contrast, RT-PCR can be performed with small volumes of plasma, which may be stored frozen for long periods without loss of DNA integrity.[30]

Ksouri, et al. have described that polymorphonuclear leukocytes (PMNLs)-based assays are more efficient in monitoring CMV reactivation, but for high-risk patients with graft-versus-host disease, more sensitive RT-PCR assays must be carried out.[31] A standardized RT-PCR assay is more appropriate than the antigen assay for detecting CMV. It allowed earlier diagnosis of active CMV infection and monitoring of the response to anti-CMV treatment.[32] The PCR detected all clinically significant CMV infections (>10 positive cells in pp65 test) and infections which required antiviral treatment. Other authors have described that the quantitative PCR appears to be a suitable alternative to diagnose and monitor CMV infections in transplant patients.[33] Therefore, we undertook this study to know the value of pp65 antigenemia using the culture technique.

Human fibroblasts have represented the conventional cell substrate for recovery of HCMV from clinical samples since the beginning of medical virology. Due to the increasing need for recovery of HCMV from the blood of immunocompromised patients, in our study, we have demonstrated the successful isolation of HCMV from human corneal fibroblast cultures. We have also demonstrated the correlation of pp65 antigenemia count of renal transplant patients and isolation of HCMV from the leukocytes of the same group.

The isolation of HCMV is not easy. As the experiment of isolation needs to be carried out with the same batch of fibroblast culture, we performed the viral isolation on 51 consecutive pp65 antigenemia-positive peripheral blood specimens. We would also like to state that the rate of isolation in the manuscript is 41.17% using the human fibroblast cell culture. In a study conducted by Veal et al.,[34] the HCMV isolation rate was 16.9%. In another study, Mazzulli et al. compared the isolation of HCMV and pp65 antigenemia assay and reported an isolation rate of 40.89%. The HCMV isolation rate reported in our study correlated with the results of Mazzulli et al.[35]

Human corneal fibroblast cultures have proven to be the best substrate for the isolation of the virus in this study. It has been proved that HCMV can be isolated from 42.85% of the pp65 antigenemia-positive individuals, but in our laboratory setup the cutoff value was 23 cells /2 × 10⁵ cells.

From the literature it is evident that the cutoff pp65 value of 10 is considered to be significant in renal transplant patients,[333637] except a report made by Gerna et al. in which it is suggested that a pp65 antigenemia count of 100 was considered significant.[38] In India, the seroprevalence of HCMV is reported to be very high among normal population and renal transplant patients.[39–41] In our earlier report, we reported the rate of positivity of HCMV to be very high among the transplant patients.[27] Therefore a pp65 antigenemia count of 10 reported based on the Western literature[42–46] may not reflect the true situation of replication of HCMV in PBLs in Indian patients. It should also be borne in mind that pp65 antigenemia assay is subjective as it is prone to the visual errors of individuals reporting on the smears. Therefore, we attempted to estimate the significant pp65 antigenemia count that would have viable HCMV in the peripheral blood.

Despite the fact that pp65 antigenemia is the most valuable tool in the diagnosis of HCMV infections, the viral isolation has also been a useful tool in diagnostics of HCMV disease.

In conclusion, HCMV was successfully cultivated for the first time in India on human corneal fibroblast culture. A pp65 count of 23 was determined to be the value of clinical significance as it gives culture positive results among renal transplant patients.