Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, ...analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quantification of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.
Four human cytomegalovirus (HCMV) isolates from four different AIDS patients treated with both ganciclovir and foscarnet and not responding clinically to antiviral treatment, were studied in order to ...verify the occurrence of double resistance to both drugs, and to define whether single or multiple HCMV strains could be responsible for the double resistance. Peripheral blood leukocytes (PBL), the relevant conventional viral isolates, and plaque-purified strains from all four patients were examined by antiviral drug susceptibility testing by an immediate-early antigen plaque reduction assay and by restriction fragment length polymorphism (RFLP) analysis using polymerase chain reaction (PCR)-amplified multiple genome regions and endonucleases. All four HCMV strains had a high level of resistance to both ganciclovir and foscarnet. A single HCMV strain was shown to be responsible for the dual resistance in each patient. HCMV strain identity and uniqueness were shown for each of the four patients in blood samples, viral isolates, and plaque-purified strains. In addition, in two patients the same single HCMV strain shifted progressively from drug sensitivity to ganciclovir and then to ganciclovir-foscarnet resistance. These findings document that resistance to both ganciclovir and foscarnet of HCMV strains recovered from blood of AIDS patients represents an emerging problem. Although it is known that multiple HCMV strains may cocirculate in the blood of AIDS patients, single strains appear to be responsible for the dual resistance. Molecular mechanisms responsible for the double resistance of the four reported strains are under study.
Seven AIDS patients with central nervous system (CNS) disease and cytomegalovirus (CMV) DNA in cerebrospinal fluid (CSF) were evaluated before and 3 weeks after standard ganciclovir treatment by ...nested polymerase chain reaction (PCR) on limiting dilutions and by quantitative PCR. After therapy, PCR CSF was negative for CMV in 3 patients with low baseline levels of CMV DNA and positive with decreased DNA titers in 4 patients with higher baseline levels. CMV pp65 antigen in polymorphonuclear leukocytes was found in 6 of 7 patients before therapy and in none of 5 after therapy. At autopsy, CMV was found in the CNS of the 4 cases examined, including 3 whose CSF was continuously positive by PCR. Quantitative PCR of CSF is useful for monitoring ganciclovir treatment in CMV infection of the CNS. In AIDS patients, standard ganciclovir treatment seems effective in reducing but not suppressing viral replication in severe cases of CMV infection of the CNS.
The reduction in the efficacy of rescue treatment (administered on a clinical basis) due to drug resistance was retrospectively quantified in 55 human immunodeficiency virus type 1 (HIV-1)-infected ...patients failing highly active antiretroviral therapy (HAART) by using a novel score calculation system based upon HIV-1 reverse transcriptase (RT) and protease (PR) mutations. Patients were all naive for nelfinavir (NFV) and efavirenz (EFV) and were assigned to one of the following rescue therapy schedules: (i) 17 patients received NFV+EFV+stavudine (d4T) (group A); (ii) 14 patients received NFV+saquinavir (SQV)+lamivudine (3TC)+d4T/zidovudine (AZT) (group B); (iii) 19 patients received NFV+d4T+didanosine (ddI)/3TC/zalcitabine (ddC) (group C); (iv) five patients received miscellaneous treatments including NFV (group D). Responders were considered patients showing a drop in HIV-1 RNA level>0.5
log
10 after 3 months of therapy. Forty-eight (28 responders and 20 non-responders) out of 55 patients completed the first 3 months of rescue therapy and reduction in HIV-1 viral load was found to be significantly higher in group A compared to groups B and C (81.2% responders vs. 38.5 and 40.0%, respectively). At baseline, no patient carried EFV- or d4T-resistant HIV-1 strains, despite prolonged administration of d4T, while 41/48 (87.2%) patients had mutations conferring resistance to NFV in the absence of previous treatment with this drug. A significant inverse correlation between reduction in viral load and reduction in therapy efficacy due to drug resistance, as determined by the score calculation system, was found (
r=0.62). A cut-off value of 36% reduction in therapy efficacy showed a positive predictive value (capacity to detect failure of rescue treatment) of 81.2% and a negative predictive value (ability to detect successful treatment) of 75.8%. In addition, 45 out of 48 patients completed also the 9–12 month period of rescue therapy and 10/28 responders had a rebound in HIV-1 viral load level detected after the first 3 months of rescue therapy. Of these, 5/7 (71.4%) showed a further reduction in rescue therapy efficacy due to the emergence of new mutations.
Background: Quantification of viral load in blood has proven to be helpful in the follow-up of disseminated HCMV infections in immunocompromised patients.
Objectives: (i) To describe the antigenemia ...assay and its optimization and (ii) to analyze the use of the antigenemia assay for the diagnosis and monitoring of HCMV infections and for the detection of treatment failures.
Study design: This article is intended to give an overview of our experience in the use of the antigenemia assay.
Results and conclusions: In solid organ transplant recipients and patients with AIDS, HCMV symptomatic infections mostly appear when antigenemia values are >300 pp65-positive PBL/2×10
5 examined. To avoid the appearance of overt HCMV disease antiviral treatment could be administered when antigenemia levels are >100 pp65-positive PBL/2×10
5 examined. Bone marrow transplant recipients show symptomatic HCMV infections when antigenemia values are >100 pp65-positive PBL/2×10
5 examined. This group of patients should be treated when antigenemia levels are <10 pp65-positive PBL/2×10
5 examined due to the higher mortality rate associated with HCMV complications. A decrease in antigenemia levels during therapy indicates a good response to antiviral drug, while stable or increasing values document a treatment failure and the emergence of drug-resistant HCMV strains.
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