P. D. Griffiths

Symptomatic cytomegalovirus infection in seropositive kidney recipients: reinfection with donor virus rather than reactivation of recipient virus.

74 patients receiving cadaver kidney grafts were investigated prospectively for cytomegalovirus (CMV) infection. Among seropositive recipients CMV infection, especially symptomatic and disseminated infection, occurred significantly more frequently when kidneys came from seropositive than from seronegative donors. Since seropositive recipients can become infected with donor virus, the excess is probably accounted for by reinfection. This conclusion was supported by restriction enzyme typing of virus isolates from recipient pairs receiving kidneys from the same donor; proven reinfection with donor strain virus was significantly commoner than proven reactivation of recipient virus. Furthermore, symptoms occurred only in the proven reinfection group. Although the proportion of reinfections that caused symptoms was less than that seen in primary infections, prior natural infection with CMV clearly does not prevent symptomatic reinfection in seropositive recipients, a point which has profound implications for future vaccination strategies in renal allograft recipients and choice of donors.

IS CYTOMEGALOVIRUS INTERSTITIAL PNEUMONITIS IN TRANSPLANT RECIPIENTS AN IMMUNOPATHOLOGICAL CONDITION

The conventional explanation for the high fatality rate due to cytomegalovirus (CMV) pneumonitis among allogeneic transplant recipients is that immunosuppression renders the host unable to control replication of this opportunistic agent. However, evidence from studies in man and the murine model of CMV show that virus replication in the lung is unrelated to the development of pathological effects, and that a host immune response is required for the induction of pneumonitis. Thus the hypothesis is that limited CMV replication in the lungs leads to display of a virus-coded protein, which is recognised by host T-cells, and that the pneumonitis is due to an uncontrolled accumulation and recruitment of such cells in the lungs. The reason why CMV is found in the lungs of patients with the acquired immunodeficiency syndrome (AIDS) without producing pneumonitis is probably because these patients cannot mount the pathogenic T-cell response. According to the hypothesis stated here, if the immune capabilities of AIDS patients can be restored, life-threatening CMV pneumonitis may develop.

CYTOMEGALOVIRUS INFECTION AND PROGRESSION TOWARDS AIDS IN HAEMOPHILIACS WITH HUMAN IMMUNODEFICIENCY VIRUS INFECTION

To examine whether cytomegalovirus (CMV) infection could accelerate progression of human immunodeficiency virus (HIV) infection to AIDS, serological studies were done on 108 HIV-infected haemophiliacs. In the 1.3-9 years from time of first recognised HIV seroconversion, the age-adjusted risk of CDC group IV disease in CMV-seropositive patients was 2.5 times that in CMV-seronegative patients. CMV-seropositive patients were also more likely to have detectable p24 antigenaemia. Survival analysis showed that CMV-seropositive patients were at greater risk of HIV disease than CMV-seronegative patients from about 2 years after HIV seroconversion. Thus CMV infection is associated with a more rapid progression to HIV disease.

Interrelationships among Quantity of Human Cytomegalovirus (HCMV) DNA in Blood, Donor-Recipient Serostatus, and Administration of Methylprednisolone as Risk Factors for HCMV Disease following Liver Transplantation

Longitudinal analysis of 162 liver transplant recipients identified 51 patients who were viremic. Virus load was determined in 47 of these patients using quantitative-competitive polymerase chain reaction. Peak virus load was significantly higher in 20 symptomatic patients than 27 asymptomatic patients (P < .0001). Elevated virus load, donor seropositivity, and total methylprednisolone dosage were risk factors for human cytomegalovirus (HCMV) disease (odds ratio [OR], 2.22/0.25 log10 increase in virus load, P = .001; OR, 4.11, P = .05; OR, 1.30/1-g increment in methylprednisolone, P = .01). Methylprednisolone and virus load were independent risk factors in a multivariate analysis (OR, 2.70/1-g increase, P = .003; OR, 1.61/0.25 log10 increase, P = .03, respectively). Virus loads of 10(4.75)-10(5.25) genomes/mL of blood were associated with an increased disease probability; the latter was shifted to lower virus loads with increasing quantities of methylprednisolone. These data illustrate the central role of virus load in HCMV pathogenesis.

Longitudinal fluctuations in cytomegalovirus load in bone marrow transplant patients: relationship between peak virus load, donor/recipient serostatus, acute GVHD and CMV disease

Quantitative competitive PCR was used to monitor the quantity of cytomegalovirus (HCMV) in 1647 blood samples from 110 BMT recipients. DNAemia was detected in 49/110 (45%) of the patients, of whom 15/49 experienced HCMV disease. Peak virus load during surveillance was elevated in symptomatic (median 4.5 log10 genomes/ml) vs asymptomatic patients (median 3.6 log10 genomes/ml, P = 0.002) and was also significantly elevated in HCMV seropositive recipients of seronegative marrow, (R+D−, median 5.0 log10), compared to those in the R−D− and R+D+ groups (P < 0.01 and <0.005). odds ratios for disease per 0.25 log10 increase in viral load, recipient seropositivity and aGVHD were 1.43 (P = 0.004), 6.60 (P = 0.05) and 3.17 (P = 0.08), respectively. In multivariate logistic regression analysis only elevated viral load remained a significant risk factor for HCMV disease. The computed disease probability viral load curve showed a rapid increase in disease risk at viral loads between 3.8 and 5.5 log10 genomes/ml in blood, and odds ratios for disease were determined for different threshold viral loads. These data demonstrate the central role of viral load in the pathogenesis of HCMV in BMT recipients and provide an additional marker for targeting and monitoring therapy.

Cytomegalovirus (CMV) viraemia detected by polymerase chain reaction identifies a group of HIV-positive patients at high risk of CMV disease

Background: Cytomegalovirus (CMV) disease is a major cause of morbidity in patients with HIV infection. Despite treatment, CMV retinitis causes substantial visual loss, especially in patients with CD4 cell counts below 50 × 106/l. Although routine ophthalmological screening of these patients has been recommended, no controlled trials have evaluated how frequently it should be performed. The aim of this study was to assess whether CMV polymerase chain reaction (PCR) results could direct ophthalmological screening to patients at high risk of CMV retinitis. Methods: In a prospective study of HIV‐positive patients with CD4 cell counts below 50 × 106/l, CMV viraemia was detected by qualitative PCR of whole blood. Patients who were CMV PCR‐viraemic were allocated to monthly virological and ophthalmological follow‐up; patients who were PCR‐negative received 3‐monthly virological and ophthalmological follow‐up. CMV viral load was determined in all CMV‐positive samples using a quantitative competitive PCR. Results: Nineteen out of 97 patients developed CMV disease over the first 12 months of the study. Sixteen (59%) out of 27 patients who were CMV‐positive developed disease compared with three (4%) out of 70 of patients who were PCR‐negative (P = 0.0001). A positive CMV PCR result was significantly associated with the development of disease (P= 0.0001), with a relative hazard of 20.15 [95% confidence interval (CI), 5.80–69.98]. Median CMV viral load was significantly higher in those individuals who went on to develop CMV disease (P = 0.02). In PCR‐positive patients, each 0.25 log10 increase in viral load increased the risk of disease (relative hazard, 1.37; 95% CI, 1.15–1.63; P = 0.0004). Conclusions: CMV PCR predicts the development of CMV disease and can be used to target ophthalmological resources to those patients at highest risk of retinitis. Asymptomatic patients who are PCR‐positive represent a high‐risk group in whom controlled trials of pre‐emptive therapy could be conducted.

TRANSFER OF A FUNCTIONING HUMORAL IMMUNE SYSTEM IN TRANSPLANTATION OF T-LYMPHOCYTE-DEPLETED BONE MARROW

To test the effect of transplantation of T-cell-depleted bone marrow on recipient immune function the results of pre-transplantation immunisation with tetanus toxoid and hepatitis-B vaccine were studied in 38 donor-recipient pairs. Immunisation of the donor alone resulted in transfer of an antibody response to the recipient; immunisation of both donor and recipient resulted in potentiation of the antibody response in both magnitude and duration. These findings indicate that donor T-cell-depleted marrow can transfer humoral immunity to the recipient and that appropriate pre-transplant immunisation schedules may be of benefit to the recipient.

Prospective study of human betaherpesviruses after renal transplantation - Association of human herpesvirus 7 and cytomegalovirus co-infection with cytomegalovirus disease and increased rejection

BACKGROUND Human herpesvirus 6 (HHV-6) and HHV-7 are two lymphotropic herpesviruses, which, like cytomegalovirus (CMV), have the potential to be pathogenic in immunocompromised individuals. We have conducted a prospective investigation to compare the natural history of HHV-6 and HHV-7 infection with that of CMV after renal transplantation. METHODS Polymerase chain reaction was used to identify infections and quantify the viral load of CMV, HHV-6, and HHV-7 in peripheral blood samples from 52 renal transplant recipients. Betaherpesvirus infections were related to defined clinical criteria obtained by detailed examination of the clinical records of each patient for the immediate 120-day posttransplant period. RESULTS CMV was the most commonly detected virus after transplant (58% of patients), followed by HHV-7 (46%) and HHV-6 (23%). Examining the time to first polymerase chain reaction positivity, HHV-7 infection was detected earlier than CMV (P=0.05). The median maximum CMV viral load was significantly higher than those for HHV-6 (P=0.01) and HHV-7 (P<0.0001) and a trend for HHV-7 viral load to be greater than HHV-6 (P=0.08). Clinicopathological analyses revealed that, in those patients with rejection, HHV-7 was associated with more episodes of rejection (P=0.02). In addition, there was a significant increase in CMV disease occurring in patients with CMV and HHV-7 co-infection compared to those with CMV infection only (P=0.04). CONCLUSIONS HHV-7 should be further investigated as a possible co-factor in the development of CMV disease in renal transplant patients and may potentially exacerbate graft rejection. No clear pathological role was observed for HHV-6.

Beta 2 microglobulin enhances the infectivity of cytomegalovirus and when bound to the virus enables class I HLA molecules to be used as a virus receptor.

We have previously demonstrated that human cytomegalovirus (CMV) binds the host protein beta 2 microglobulin (beta 2m) from body fluids or from cell culture media. In this report we have examined the effect of the beta 2m on viral infectivity. We have shown that the addition of human purified beta 2m, or a fraction of foetal calf serum corresponding to bovine beta 2m, to culture medium increased the amount of infectious extracellular CMV, compared to that from cells grown in serum-free medium. Metabolic labelling experiments demonstrated that this effect was not due to an increase in the amount of extracellular virus but to an increase in the infectivity of the virus present in extracellular fluids. We concluded that the binding of beta 2m by CMV increased its infectivity. We have shown that CMV and beta 2m compete for binding sites on fibroblasts. As the main binding site on cells for beta 2m is the class I HLA heavy chain we compared the binding of CMV to the Raji and Daudi cell lines which express or lack expression of class I HLA molecules. The binding of radiolabelled beta 2m-coated CMV was significantly higher to Raji cells than to Daudi cells. Furthermore, CMV could compete with beta 2m for binding to Raji cells, although the reverse was not true. These results demonstrate that CMV can use class I HLA molecules as a virus receptor. We propose that when coated with beta 2m, CMV has the capacity to displace beta 2m from the class I HLA heavy chain-beta 2m dimer on the cell surface and bind to cells. The fact that beta 2m enhances infectivity suggests that such binding leads to productive infection of cells.

LONGITUDINAL ANALYSIS OF CYTOMEGALOVIRUS LOAD IN RENAL TRANSPLANT RECIPIENTS USING A QUANTITATIVE POLYMERASE CHAIN REACTION : CORRELATION WITH DISEASE

Serial surveillance samples of urine collected from 103 renal transplant recipients were analysed by polymerase chain reaction (PCR) for the presence of human cytomegalovirus (HCMV) DNA. The PCR results were consistently negative in 70 patients, none of whom developed HCMV disease, and PCR positive in 33 patients of whom 10 developed HCMV disease (P < 0.001). In 12 patients, PCR results were positive in three or more consecutive samples indicating extensive HCMV replication. HCMV load in 104 samples from these patients was analysed using a quantitative co-amplification PCR system. The maximal viral burden in the symptomatic patients ranged from 10(5.9) to 10(7.12) genomes/ml urine (median 10(6.5)) and in the asymptomatic patients from 10(4) to 10(5.7) genomes/ml urine (median 10(5.2)). The 10(1.3) difference between these median values was significant (P < 0.01). Individual kinetic profiles of viral burden showed that high levels of HCMV correlated with clinically apparent disease. In the majority of the asymptomatic individuals HCMV load remained between 10(4) and 10(5.1) genomes/ml urine; however, in two patients fluctuations in viral load were observed involving higher viral levels (up to 10(5.7) genomes/ml urine) suggesting that immune responses able to modulate viral replication could be studied in individual patients. Analysis of the temporal appearance and quantity of HCMV in the urine with alterations in white cell numbers showed that leukopenia occurred following the appearance of HCMV in the urine of symptomatic patients but preceded HCMV in the urine of asymptomatic patients (P = 0.01). Overall, these results show that longitudinal analysis using fully quantitative PCR methods for HCMV can provide insight into the natural history of HCMV disease in renal transplant recipients.