Jana Lochmanová

Cytomegalovirus encephalitis/retinitis in allogeneic haematopoietic stem cell transplant recipient treated successfully with combination of cidofovir and foscarnet.

Abstract:  We report an 18‐yr‐old female patient with repeated CMV reactivations after HSCT treated by several pre‐emptive courses of virostatic therapy. Seven months after HSCT, she developed CMV encephalitis/retinitis. Initial therapy with GCV and hyperimmune globulin failed, and later on GCV‐resistant strain was detected. Continual increase of CMV DNA in peripheral blood led us to combined therapy with CDV and FCV, which was successful and free of severe renal toxicity. To our best knowlegde, this is the first reported case of successful CMV treatment with a combination of CDV and FCV.

Real-Time PCR Diagnostics Failure Caused by Nucleotide Variability within Exon 4 of the Human Cytomegalovirus Major Immediate-Early Gene

ABSTRACT Here we report how variability in the human cytomegalovirus genome sequence may seriously affect the outcome of its molecular diagnosis. A real-time quantitative PCR assay targeting the major immediate-early gene failed to detect the viral load in some, but not all, clinical samples from hematooncological patients. By amplification and sequencing the DNA across the regions targeted by this assay we found a number of nucleotide substitutions which accounted for decreased primer/probe binding. This decreased the sensitivity of the assay up to 1,000-fold.

Detecting human cytomegalovirus drug resistant mutations and monitoring the emergence of resistant strains using real-time PCR

BACKGROUND Antiviral resistance development is a serious complication of human cytomegalovirus virostatic therapy caused by mutations in UL 97 and/or UL54 genes. OBJECTIVES To determinate the presence of sensitive and resistant strains in patients developing antiviral resistance. STUDY DESIGN We used three different molecular biological methods for mutation analysis-restriction fragment length polymorphism, sequencing and real-time PCR approach. RESULTS We describe three allogeneic hematopoietic stem cell transplant patients developing the GCV resistant HCMV strains manifested by virostatic treatment failure. In these patients we identified UL97 mutations L595S, A594V and A594T and monitored the dynamics of coexisted sensitive/resistant strains. We confirmed the presence of mixed HCMV populations and in two patients a phenomenon of sensitive strain repopulation which occurred after 6.5 months and 1 month after removing GCV pressure. CONCLUSIONS Our results show changes in proportions of sensitive/resistant subpopulations over time but other studies would be required to demonstrate the beneficial impact of their monitoring on clinical outcome.

Specific p53 mutations do not impact results of alemtuzumab therapy among patients with chronic lymphocytic leukemia

A poor prognosis in chronic lymphocytic leukemia (CLL) is associated particularly with the presence of del(17p) aff ecting tumor suppressor gene TP53 . Th is deletion is in almost all cases of progressive leukemia accompanied by a mutation in the other TP53 allele, and in a smaller proportion of patients the TP53 mutation occurs also independently of del(17p) [1]. Recently, we demonstrated that patients with CLL harboring a missense mutation located in the p53 DNA-binding motifs (DBMs) (structurally well-defi ned parts of the DNA-binding domain) manifested a clearly shorter median survival in comparison with those having a missense mutation outside DBMs or a non-missense alteration [2]. However, a limitation of this study resulted from the unpredictable survival impact of diverse therapy given to patients with p53 mutations. Th e therapeutic approach currently taken for patients with CLL with loss and/or mutation of TP53 relies mostly on the use of agents which do not act through DNA damage followed by apoptosis induction. Th e success of this approach has been documented for the monoclonal antibody alemtuzumab. Monotherapy with alemtuzumab is now being recommended as fi rst-line therapy for patients with CLL with del(17p) [3]; however, many unresolved questions need to be addressed. For example, it is unclear whether the type of p53 mutation infl uences the outcome of alemtuzumab therapy. In an eff ort to evaluate the effi cacy of alemtuzumab therapy in relation to specifi c p53 mutations in patients with CLL, we performed a retrospective analysis. Th e patients examined ( n 111) were treated by alemtuzumab between 2003 and 2010. Th e majority of cases concerned pretreated patients ( n 108; see Table I). Our present analysis was performed in the patient cohort which was largely included in the previous survival analysis related to p53 mutation types; this study also involved patients treated with other drugs in addition to alemtuzumab [2] (overlap 88% in group 1; 82% in group 2; 0% in group 3; the groups are defi ned further below). Defects in p53 were assessed by the yeast functional assay coupled to sequencing of DNA templates from mutated yeast colonies. Th e remaining TP53 allele was analyzed using