Viral Infections

Screening for cytomegalovirus (CMV) infection in allogeneic bone marrow transplantation using a quantitative whole blood polymerase chain reaction (PCR) method: analysis of potential risk factors for CMV infection

Abstract

Potential risk factors for CMV infection and the use of quantitative CMV PCR screening to guide pre-emptive anti-CMV therapy were reviewed retrospectively in 32 allogeneic bone marrow transplant patients accrued over a 2-year period. Significant CMV PCR positivity (an indicator of CMV infection) developed in 34% of patients. When analysed by recipient CMV IgG serostatus, 69% of seropositive recipients developed significant CMV PCR positivity while none of the seronegative recipients did so (P = 0.00007). Considering only the seropositive recipients, 100% of those who received the low intensity campath-1H/fludarabine/melphalan ‘mini-allograft’ conditioning regimen developed significant CMV PCR positivity, while only 44% of those who had received cyclophosphamide/TBI did so (P = 0.0337). The mean time to first episode of significant CMV PCR positivity for those who had received campath/fludarabine/ melphalan was 25 days while for those who had received cyclophosphamide/TBI, this was 66 days (P = 0.0372). For the first episode of significant CMV PCR positivity, the mean index and peak CMV PCR counts for those who had received campath/fludarabine/melphalan were 4.54 and 5.22 log copies/ml respectively, while for cyclophosphamide/TBI, the corresponding figures were 3.85 and 4.12 log copies/ml respectively (P = 0.2986 and P = 0.0472 for index and peak values). 85% of those who had significant CMV PCR positivity with the campath/fludarabine/melphalan regimen developed more than one such episode, while 50% of those receiving cyclophosphamide/TBI regimen did so (P = 0.491). Significant CMV PCR positivity was associated with symptoms in a proportion of patients (pyrexia 45%, cough 18%, rise in AST 72%). No patient developed overt CMV disease. CMV PCR is useful for guiding pre-emptive anti-CMV therapy and for monitoring response. Bone Marrow Transplantation (2001) 27, 301–306.

Main

Increasing numbers of patients are undergoing allogeneic bone marrow transplantation for haematological malignancies and other conditions. While antibacterial and antifungal prophylaxis and therapy have arguably limited classical sepsis, the prevention of cytomegalovirus (CMV) infection and disease has been more problematic. The development of cytomegalovirus infection and disease in bone marrow transplant patients may result in severe complications such as pneumonitis, which may prove fatal.1

A number of strategies have been devised to allow intervention before the onset of CMV disease, and have been extensively reviewed elsewhere.2345678 These have included the use of aciclovir or ganciclovir, either orally or intravenously as primary prophylaxis. An alternative approach has been to offer pre-emptive full dose anti-CMV therapy (usually ganciclovir) to those patients developing positivity in markers for CMV infection, the rationale being to limit therapy related side-effects, such as neutropenia which may increase mortality,9 while maximising the benefit of therapy for selected high risk patients in preventing CMV disease. Initial screening methods involved the detection of CMV antigenaemia or the culture of virus from blood or respiratory fluids. More recently, PCR based screening methods for the detection of CMV infection in allogeneic bone marrow transplant patients have been adopted in some centres.

Patients and methods

Since 1 January 1998, patients undergoing allogeneic bone marrow transplantation at the Christie Hospital have been screened weekly by quantitative whole blood CMV PCR, from the week preceding transplantation to 100 days post-transplantation and then fortnightly until stopping cyclosporin. CMV PCR may also be performed at any time on clinical indications. Those patients developing significant CMV PCR positivity had further CMV PCR tests performed at less than weekly intervals to monitor PCR counts and response to pre-emptive therapy with daily ganciclovir i.v. (usually 5 mg/kg twice daily), which was given for at least 10 to 14 days and until the CMV PCR test had become consistently negative. These patients were then started on a maintenance (prophylactic) regimen of ganciclovir i.v. (5 mg/kg per dose) twice or three times weekly, which was generally continued until at least 100 days post transplantation. If patients developed further episodes of significant PCR positivity, daily ganciclovir was restarted and patients managed as above. Foscarnet therapy and viral sensitivity testing were considered in those patients failing to respond, in terms of PCR counts, to ganciclovir.

Additionally, all patients received aciclovir 200 mg p.o. four times daily as prophylaxis against herpes simplex, and this was started on the 3rd or 6th day pre-transplant and continued until 6 weeks post transplantation. All CMV seronegative recipients received CMV seronegative blood or blood products. All patients received specialist medical care including the use of G-CSF and immunoglobulin as relevant to their conditioning regimen and as required subsequently. Appropriate antifungal, antibacterial and antiprotozoal prophylaxis was used. Patients were nursed in a dedicated HEPA filtered facility with appropriate isolation protocols.

Conditioning regimens used in the transplant recipients were as follows: campath-1H/fludarabine/melphalan (a low intensity regimen10) or cyclophosphamide 60 mg/m2/TBI 1200 Gy or campath/cyclophosphamide/TBI or busulphan/melphalan. GVHD prophylaxis consisted of either cyclosporin/methotrexate or cyclosporin alone.

In this retrospective study, data from all patients who had undergone allogeneic bone marrow transplantation between 1 January 1998 and 31 December 1999 were reviewed with regard to CMV infection. Case notes of those patients who had developed significant CMV PCR positivity following their transplant were reviewed and relationships between significant CMV PCR positivity, associated symptoms and immunosuppressive regimens were investigated.

Quantitative CMV PCR was performed on EDTA whole blood samples. Briefly, DNA was extracted from 1 ml volumes using the Nucleon blood extraction kit BCC1 (Nucleon Biosciences, Glasgow, UK) and amplified using a previously described Taqman quantitative CMV PCR assay format.11 The limit of detection for this assay was defined as 500 CMV DNA genome copies/ml. CMV PCR data on the study patients were obtained from the virology computer database. Significant CMV PCR positivity was defined by the development of a positive PCR result, with subsequent positive results on retesting the patient within the next 24 to 36 h.

Neutropenia was defined below an absolute count of 0.5 × 109/l. Graft-versus-host disease status was defined on documented clinical findings or biopsy results. Symptoms of cough were identified from the medical notes. Pyrexia was defined as a temperature of 37.5°C or greater. Subjectively, a rising trend in aspartate aminotransferase (AST) was judged to be associated with CMV PCR positivity if it immediately preceded or coincided with the onset of a period of significant CMV PCR positivity.

Statistical inferences were made using the two-sided Fisher's exact test for categorical data and the two-sided Mann–Whitney U test (adjusted for ties) for numerical data.

Results

Thirty-five patients had undergone allogeneic bone marrow transplantation at the Christie Hospital between 1 January 1998 and 31 December 1999. Of these, two patients dying within 1 month of transplantation were excluded. One of these excluded patients died from probable CMV pneumonitis 9 days after transplantation, but had evidence of CMV infection pre-dating bone marrow transplantation. This patient was therefore considered ineligible for inclusion in this study on CMV PCR screening and pre-emptive therapy. A further patient who received ganciclovir as primary prophylaxis was also excluded, leaving a total of 32 study patients, the baseline characteristics of whom are outlined in Table 1.

Table 1  Characteristics of the study patients

The analysis of potential risk factors for the development of significant CMV PCR positivity is outlined in Table 2. While significant CMV PCR positivity developed in 11 out of the 32 patients (34%), this risk differed markedly depending on the pre-transplant CMV IgG serostatus of recipient and donor: Recipient+/donor+ 60%, recipient+/donor− 83%, recipient-/donor+ 0%, recipient−/donor− 0%. Overall, 69% (11 out of 16) of CMV seropositive recipients developed significant CMV PCR positivity while none (0 out of 16) of the CMV seronegative recipients did so (P = 0.000068). Considering only the seropositive recipients, as all cases of significant CMV PCR positivity were from this subgroup of 16 patients, 100% (seven out of seven) of those receiving campath/fludarabine/melphalan developed significant CMV PCR positivity, while only 44% (four out of nine) of those who had received cyclophosphamide/TBI did so (P = 0.0337). No seropositive patient in our study had received any other regimens.

Table 2  Analysis of potential risk factors for the development of significant CMV PCR positivity

Demographic and risk factor data for the 11 patients who developed significant CMV PCR positivity are outlined in Table 3. The mean age for these patients was 45 years (range 25–56 years) and 55% were males. Seven of these patients (64%) developed graft-versus-host disease, of whom four had an onset before and three an onset after the development of significant CMV PCR positivity. None of the patients had neutropenia concurrent with or within the preceding 48 h of any CMV PCR positive period, while three patients (27%) had been on concurrent systemic steroids or had received them within the preceding 48 h of any CMV PCR positive period.

Table 3  Demographic and risk factor data for patients who developed significant CMV PCR positivity

CMV PCR positivity data for the 11 patients who developed significant CMV PCR positivity are outlined in Table 4. The mean time from transplantation to significant CMV PCR positivity for all 11 patients was 40 days (range, 5 to 134 days). Of these patients, the mean time to positivity for those who had received campath/ fludarabine/melphalan (seven patients) was 25 days (range, 5–36 days), while for those who had received cyclophosphamide/TBI (remaining four patients), the mean time to positivity was 66 days (range, 33–134). This difference was statistically significant (P = 0.0372). For all 11 patients, the mean index and peak CMV PCR counts for the first episode of significant CMV positivity were 4.40 (range, 2.96–5.00) and 5.01 (range, 3.92–5.55) log copies/ml respectively. Of these 11 patients, the mean index and peak counts for those who had received campath/fludarabine/melphalan were 4.54 (range, 3.00–5.00) and 5.22 (range, 3.98–5.55) log copies/ml respectively, while for those who had received cyclophosphamide/TBI, the corresponding figures were 3.85 (range, 2.96–4.15) and 4.12 (range, 3.92–4.39) log copies/ml respectively (P = 0.2986 and P = 0.0472 for index and peak values, respectively). More than one episode of CMV PCR positivity occurred in eight of the 11 patients (72%). When this was analysed by regimen received, 100% of patients (seven out of seven) who had significant CMV PCR positivity with the campath/fludarabine/melphalan regimen developed more than one such episode, while 50% of patients (two out of four) with the cyclophosphamide/TBI regimen did so (P = 0.109).

Table 4  CMV PCR data and associated symptoms for patients who developed significant CMV PCR positivity

Symptoms associated with significant CMV PCR positivity are also outlined in Table 4. Associated fever was noted in five of the 11 patients (45%). Associated cough was noted in two patients (18%). A rising trend in AST associated with development of CMV PCR positivity was noted in eight patients (72%). No patient developed overt CMV disease.

Most patients with significant CMV PCR positive episodes responded promptly to intravenous ganciclovir therapy, with CMV PCR counts falling on commencement of full dose ganciclovir. A time course for one of these patients is outlined in Figure 1. However, two patients required a change from intravenous ganciclovir to intravenous foscarnet to drop CMV PCR counts.

Figure 1
figure1

Time course of CMV PCR counts for patient 6. GCV = ganciclovir.

Discussion

Our study indicates that a positive CMV serostatus in the recipient is the principal risk factor for the development of CMV infection in allogeneic bone marrow transplantation, as detected by the development of significant CMV PCR positivity. In our series, no seronegative recipient developed CMV infection, whatever the donor serostatus and conditioning regimen. Seropositive recipients receiving the campath/fludarabine/melphalan ‘mini-allograft’ regimen may be at particular risk of developing CMV infection, and in our study all seropositive recipients who had received this regimen developed significant CMV PCR positivity. In non-transplant patients, fludarabine regimens have been used in the treatment of chronic lymphocytic leukaemia, but an increase in CMV infection has not been noted.12 While both campath and fludarabine have be associated with CMV infection,1314 campath may be the more potent risk factor. These agents are likely to cause significant impairments in cell-mediated immunity and their use in combination may result in an even greater vulnerability to CMV infection.15

While no patient in our study developed overt CMV disease, the development of significant CMV PCR positivity was associated with symptoms consistent with infection in a significant proportion of patients. It is reasonable to assume that these patients would have been at significant risk of progression to overt CMV disease had pre-emptive therapy not been given. Overt CMV disease is associated with a poor outcome, as noted in the non-study patient with evidence of CMV infection predating transplantation who died from probable CMV pneumonitis 9 days following bone marrow transplantation.

Our previous CMV screening strategy in allogeneic bone marrow transplants was based on CMV antigenaemia, and data exist to support such a strategy.161718 However, CMV antigenaemia detection is a relatively labour-intensive procedure and sample processing needs to be commenced within 3 h of specimen receipt.19 Processing large numbers of specimens is therefore difficult. Additionally the interpretation of CMV antigenaemia tests is problematic, particularly if neutropenia is present, as is often the case in bone marrow transplant patients.

Data also exist to support CMV PCR guided pre-emptive therapy.20 Comparative data suggest that plasma CMV PCR is as sensitive as CMV antigenaemia detection2122 and peripheral blood leukocyte CMV PCR is more sensitive than CMV antigenaemia detection.23 In our experience, a quantitative whole blood CMV PCR method once set up has had the advantage of relatively easier sample processing and interpretation of results. Larger numbers of specimens may be conveniently processed. A quantitative CMV PCR method is particularly useful for following response to anti-CMV therapy, and may prompt a change in the treatment of patients failing to respond with a fall in CMV PCR counts. The level of CMV PCR positivity may indicate the degree of infection, and in our study, patients who developed significant CMV PCR positivity and had received the campath/fludarabine/melphalan regimen had consistently higher PCR counts than those who had received cyclophosphamide/TBI.

Defining threshold values is an important consideration for a quantitative screening method, and we did not start pre-emptive therapy on the basis of isolated PCR counts of under 1000 (3 log) copies/ml. Such isolated low counts were very occasional in our study, the majority of positive PCR results being clearly part of definite periods of significant PCR positivity. Data from a large prospective study of liver, renal, and bone marrow transplant patients suggest that initial CMV viral load and its rate of increase may correlate with the probability of developing CMV disease during CMV infection.24 Further study may be needed to define the threshold values that might better direct pre-emptive therapy or predict CMV disease in the different categories of transplantation.

Our data suggest that in allogeneic bone marrow transplant recipients, weekly screening by CMV PCR is successful in detecting CMV infection and preventing CMV disease by allowing timely pre-emptive therapy. As those seropositive recipients receiving the less conventional campath/fludarabine/melphalan ‘mini-allograft’ regimen have a very high likelihood of developing significant CMV PCR positivity, and at an earlier stage post-transplantation, twice weekly CMV PCR screening may be more prudent. While our CMV PCR based pre-emptive therapy prevented overt CMV disease in these patients, additional primary prophylaxis could prevent CMV reactivation altogether. Whether such an approach would lead to an overall and cost-effective benefit after accounting for primary prophylaxis associated side-effects deserves study. Additionally, the emergence of antiviral resistance may be a greater issue in patients receiving primary prophylaxis.

In seronegative recipients, our data suggest that the value of screening or primary prophylaxis may be limited as no case of significant CMV PCR positivity developed in this group.

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Acknowledgements

We thank the staff on the Acute Leukaemia Unit at the Christie Hospital for their assistance during this study.

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Correspondence to BA Oppenheim.

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Qamruddin, A., Oppenheim, B., Guiver, M. et al. Screening for cytomegalovirus (CMV) infection in allogeneic bone marrow transplantation using a quantitative whole blood polymerase chain reaction (PCR) method: analysis of potential risk factors for CMV infection. Bone Marrow Transplant 27, 301–306 (2001). https://doi.org/10.1038/sj.bmt.1702778

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Keywords

  • cytomegalovirus
  • PCR
  • allogeneic
  • bone marrow transplantation

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