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Polymerase chain reaction to evaluate antiviral therapy for cytomegalovirus disease

A sensitive and

specific method to monitor suppression of cytomegalovirus (CMV) replication is essential in patients treated with ganciclovir after allogeneic bone-marrow transplantation. In this study, antiviral therapy of eighteen episodes of symptomatic CMV infection in 15 such patients were followed up clinically and by virus culture and polymerase chain reaction (PCR). Clinical improvement, culture, and PCR were assessed for their ability to predict the efficacy of ganciclovir therapy in each patient. In eleven successfully treated episodes of CMV disease (disappearance of symptoms and improvement in biochemical variables) clinical improvement was associated with an effective suppression of virus replication as shown by negative culture and PCR assays of blood and urine specimens obtained after antiviral therapy. 1 patient who did not improve clinically when receiving antiviral therapy remained both culture positive and PCR positive for CMV. 6 patients with early relapse of CMV disease or who died after an initial clinical improvement were PCR positive but culture negative after termination of therapy. Demonstration of CMV in blood and urine by PCR after stopping antiviral therapy (even when culture is negative) points to incomplete suppression of virus replication. The findings show that PCR is a better predictor of the efficacy of antiviral therapy than are culture or clinical assessment.

introduction

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in bone-marrow transplant recipients.1 Antiviral therapy with ganciclovir, a nucleoside analogue, which in vitro has antiviral activity against herpesviruses 2,3 suppresses virus replication and improves CMV disease in most immunocompromised patients." The observation that after marrow transplantation both CMV infection of the gastrointestinal tract9 and CMVinterstitial pneumonia (IP) only partly responded to ganciclovir10,11 led to the introduction of combination therapy of the nucleoside analogue plus hyperimmune globulin. Such a combination has led to a pronounced reduction in the mortality of CMV - IP.12,13 Since the introduction of the new antiviral agents, there have been several attempts to establish the best treatment for CMV disease after bone-marrow transplantation. However, a serious obstacle to anti-CMV therapy is viral resistance to ganciclovir.14 Patients receiving ganciclovir have to be followed up carefully to detect treatment failures early. Only prompt changes in therapy, such as the addition or substitution of other antiviral agents (eg, interferons or

in patients with suboptimum response to further reduce the mortality of CMV disease after allogeneic bone-marrow transplantation. Alternative regimens have to be introduced when the side-effects of ganciclovir limit the application of the drug to the individual patient. Methods for a sensitive and specific monitoring of suppression of virus replication are essential for optimum treatment and for evaluation of the efficacy of other antiviral agents alone or combined to treat CMV disease. Lately, we have described the use of the polymerase chain reaction (PCR) technique for more rapid and sensitive diagnosis of CMV infection after bone-marrow transplantation compared with the commonly used virus culture technique.16 Here we report a study in which we applied PCR in addition to virus culture to assess the efficacy of antiviral therapy of CMV disease after allogeneic bone-marrow transplantation.

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Patients and methods Patients Between January, 1989,andAugust, 1990, 13 men and 2 women (median age 33 years, range 19-43) were consecutively studied after allogeneic bone-marrow transplantation. 12 patients had chronic myelogenous leukaemia (9 in chronic phase, 3 in accelerated phase [blast crisis]), 2 had acute myeloid leukaemia, and 1 had acute lymphoblastic leukaemia. All patients had graft-versus-host disease (grade 0-1,11 patient; grade 2-4,14 patients), and were anti-CMVIgG seropositive (4 patients), or had received a marrow graft from a seropositive donor (3), or both (8). They all had received leucocyte-depleted blood products unscreened for CMV and hyperimmue globulin prophylaxis." Oral acyclovir was given for herpes simplex prophylaxis at a dose of 400 mg four times a day until the 100th day after bone-marrow transplantation.

Diagnosis of CMV infection

’

After transplantation patients were followed up weekly by virus culture and PCR to detect CMV infections. Patients who had received antiviral therapy had positive blood and urine cultures for CMV and at least one feature of CMV disease. Additionally, all blood, urine, and organ biopsy specimens obtained from these patients before the start of treatment were found to be CMVpositive by PCR. CMV pneumonia was diagnosed on the basis of dyspnoea, interstitial infiltrates on chest radiograph, and a positive CMV culture of bronchial washings. Diagnosis of CMV enteritis was based on clinical and endoscopic signs of enteritis and colitis, demonstration of CMV inclusions, and antigen expression or culture of the virus from the tissue biopsy samples. Marrow aplasia due to CMV infection was demonstrated by hypoplastic marrow samples positive for CMV (culture or slot-blot hybridisation). The ADDRESSES: Department of Internal Medicine, Section of Transplantation Immunology and Immunohaematology (H. Einsele, MD, G Ehninger, MD, M. Steidle, PhD, M. Muller, H. Schmidt, MD, Prof J G Saal, MD, Prof H. D. Waller, MD, Prof C. A. Muller, MD); and Department of Virology, University of Tübingen (Prof A Vallbracht, PhD), Germany. Correspondence to Dr H. Einsele, Medizinische Klinik und Poliklinik, Otfried-Muller Strasse 10, D-7400

Tubingen, Germany.

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EVALUATION OF ANTIVIRAL THERAPY

i

CMV

I

i

i

I

I

I

cultured on human embryonic lung fibroblasts CMV was identified by monoclonal antibodies agamst the immediate early and applied 48 h after inoculation of the fibroblasts and by production of characteristic cytopathic effects. PCR was earned out as described m Methods *Only a shortened course of ganciclovir given because of severe neutropenia CMV-IP= CMV- interstitial pneumonia was

routmely

diagnosis of CMV hepatitis was based on abnormal liver function tests in association with culture-proven CMV viraemia. After antiviral therapy was started, patients were assessed once a week for the presence of CMV in blood and urine by virus culture and PCR. PCR

Amplification of a 147 bp DNA fragment between positions 1767 and 1913 of the 4th exon of the immediate early gene of the CMV strain AD 169 with primer I and II has been described elsewhere.15,18 Briefly, 100 ng of extracted DNA19 or the total amount of DNA extracted from 10 ml urine were denatured at 94°C for 5 min and specifically amplified in 50 hl reaction mixture (32 cycles; annealing and primer extension for 3 min at 66°C followed by denaturation for 1 min at 94°C). To reduce to a minimum the risk of contamination, the PCR technique was physically separated from DNA extraction, precipitation, and DNA recombinant studies. To exclude the presence of polymerase inhibitors and to test the quality of the extracted DNA, a DNA fragment of the human HLA class I genes (4th exon, 129 bp long) was amplified in all samples in parallel. Results were regarded as valid only if consistent in at least two independent experiments and if all the negative controls did not show amplification either in the agarose gel or after hybridisation. Detection of the amplification products in a 2% agarose gel or by Southern blot analysis with gamma3zP-dATP-end-labelled internal oligonucleotide has been described previously.15,20

Therapy Ganciclovir was given in a dose of 5 mg/kg twice daily. Patients with CMV-IP and CMV-enteritis received additional CMV hyperimmune globulin (0-2 g/kg per day for 5 days).

Definition of response to antiviral therapy Clinical response was defmed as improvement of symptoms, signs, and biochemical variables (liver function tests, hypoxaemia,

leucopenia, thrombocytopenia) Favourable virological response

related to CMV disease. defmed as negative culture

was

assays from all the sites analysed. Early relapse was defined by recurrence of symptoms of CMV disease and positive cultures from blood and urine less than 4 weeks after stopping antiviral therapy.

early antigen

when

Results

Eighteen episodes of CMV disease in 15 patients were treated with ganciclovir. 9 of these patients also received CMV hyperimmune globulin infusions to treat CMV-IP and CMV-enteritis. CMV disease manifested at a median of 56 days after allogeneic bone-marrow transplantation; the virus was detected substantially earlier by PCR than by culture (mean 23 days [range 4-35] vs 44 [14-76]). Evaluation of antiviral therapy

(table)

Sixteen of the eighteen episodes of symptomatic CMV infection were treated with a full course (14 days) of antiviral therapy. A shortened course (10 or 12 days) only could be given to 2 patients because they had severe neutropenia associated with bacterial or fungal infections. In 11 patients (3 after a second course of ganciclovir for early relapse of CMV disease after the first course), clinical and biochemical signs of CMV disease disappeared after ganciclovir treatment and cultures were negative. Negative PCR results in all patients after stopping antiviral therapy further confirmed the success of treatment. Virus infection did not recur during the first 4 weeks of follow-up as judged by both techniques. In 6 of these patients CMV infection recurred 2-5 months after stopping ganciclovir. In 3 patients with CMV-IP, improvement of respiratory symptoms and of radiological appearance of the lung were recorded after antiviral treament. Cultures from blood and urine became negative for CMV in these patients at the end of the treatment course and 1 week later. By contrast, the PCR assay revealed CMV in blood and urine samples after cessation of therapy in all these patients. There was a rapid reappearance of positive virus cultures from at least one site and a worsening of CMV disease in these patients after antiviral therapy was stopped. All patients died shortly thereafter. Necropsy (done in all but 1 patient after consent from relatives) yielded positive cultures from several organs, indicating systemic CMV disease. 1 patient with CMV-IP

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multiple other infectious complications after immunosuppressive therapy for severe acute graft-versushost disease and persistent severe neutropenia tolerated only 10 days of ganciclovir treatment. The patient died shortly thereafter due to generalised CMV infection. Virus persistence was demonstrated in this patient by positive PCR and virus culture assays during and after antiviral therapy. and

ganciclovir resistance. We believe that application of the PCR technique to follow the immunocompromised patient receiving antiviral therapy might allow a more sensitive evaluation of efficacy in the individual patient and, when applied in larger studies, might help to compare different antiviral strategies. REFERENCES

Relapse 3 patients had an early relapse of CMV disease after antiviral therapy was stopped, 1 of whom had been given ganciclovir for only 12 days because of severe neutropenia. These patients had initially shown a clinical response; cultures, done after the antiviral therapy was stopped, revealed no viral growth. By contrast, PCR demonstrated CMV in all the blood and urine specimens obtained from these patients. These 3 patients received a second course of antiviral treatment which was administered for the full cycle of 14 days. At the end of the second course of therapy virus replication was effectively suppressed, as evidenced by negative culture and PCR assays from all the sites analysed (table). Discussion We have shown that suppression of virus replication as judged by culture is not necessarily associated with effective antiviral therapy, and that PCR is a better predictor of the efficacy of such treatment. Firstly, PCR detected CMV substantially earlier after transplantation than did culture. Secondly, virus was detected by PCR but not by culture in blood and urine from the 3 patients with CMV-IP. In these patients, culture of necropsy samples confirmed that virus replication was incompletely suppressed. Likewise, in the 3 patients who relapsed soon after antiviral therapy was stopped, PCR, but not culture, demonstrated virus in blood and urine. Negative PCR assays of blood and urine specimens after stopping antiviral therapy (independent of the antiviral therapy introduced and of the manifestation of CMV disease) indicated successful suppression of virus replication for at least 4 weeks. That suppression of virus replication, rather than eradication, can be achieved by antiviral therapy, was evidenced by recurrence of CMV infection 2-5 months after antiviral therapy was stopped in 6 of the successfully treated patients. A negative PCR assay at the end of antiviral therapy seems to be a better marker for effective antiviral treatment than clinical improvement or negative culture, especially in patients with CMV-IP. Masking of the virus by anti-CMV antibodies might inhibit culture of the virus from patients with persistent CMV infection, because all the culture-negative patients, positive by PCR after antiviral therapy, had received high-dose CMV hyperimmune

globulin Thus, a PCR-positive assay after ganciclovir therapy, irrespective of resolution of clinical signs and symptoms and suppression of virus replication as demonstrated by culture, might be an indication that ganciclovir therapy should be continued. Maintenance therapy or, in the case of severe side-effects, therapeutic modifications by introduction either of additional antiviral agents or of haematopoietic growth factors to reduce marrow suppression should be considered in patients who are persistently PCR positive after antiviral therapy is stopped. The CMV strains cultivated from these patients should also be tested for

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