Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV- or EBV-positive; 25/93); (3) double-positive (9/93). The double-negative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.

Therapy-related myelodysplastic syndrome with monosomy 5 and 7 following successful therapy for acute promyelocytic leukemia with anthracyclines.

Myelodysplastic syndrome (MDS) in patients treated for acute promyelocytic leukemia (APL) is a rare event. We describe a patient with APL who developed MDS 40 months after entering complete remission (CR). Karyotypic analysis revealed monosomy 5 and 7, which are cytogenetic changes usually occurring after the use of alkylating agents. The patient had received only anthracyclines as potential leukemogenic drugs. A review of the literature on t-AML/MDS occurring after successful therapy for APL showed three similar cases. These observations suggest that anthracyclines may cause t-AML/MDS similar to that induced by alkylating agents.

Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples.

OBJECTIVE: To apply the polymerase chain reaction (PCR) to serum samples for the rapid diagnosis of Legionnaire's disease using the L5SL9 and L5SR93 primers designed to generate a 104-base-pair (bp) fragment from the 5S RNA gene of Legionella spp. The amplified product was detected by electrophoresis and by hybridization with the L5S-1-specific probe. METHODS: Single specimens of serum obtained from 24 patients with confirmed legionellosis, at different stages of their disease, were tested by PCR. Additionally, 10 serum samples from patients with no clinical symptoms of pneumonia and 10 samples from patients suffering from pneumonia caused by Mycoplasma pneumoniae, Coxiella burnetii or Chlamydia psittaci were also tested as controls in order to determine the specificity of the method. RESULTS: Of the 24 examined serum samples, the amplified products from 12 hybridized with the L5S-1 probe (sensitivity 50%). None of the negative controls was positive after PCR. No correlation was found between the day of illness and the positivity in the test. CONCLUSIONS: The PCR technique could be applied as a diagnostic tool for the rapid diagnosis of legionellosis in serum samples after modification, mainly to improve its sensitivity.

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates.

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.

Characteristics of coagulase-negative staphylococci from infants with bacteremia.

Twenty-nine infants were identified as having coagulase-negative staphylococcal (C-S) bacteremia. Fourteen infants had pneumonia and 10 had central line-associated bacteremia. Twenty-four of 29 (83%) had invasion of the mucocutaneous barrier at the time the positive blood culture was drawn. Clinical signs and symptoms were nonspecific. Apnea/bradycardia was the most prevalent clinical feature, occurring in 20 (69%) infants. Staphylococcus epidermidis was the most frequent blood culture isolate, occurring in 21 (72%) cases. Slime production by C-S blood culture isolates occurred in 23 (79%) cases. There was no prevalent antibiotic resistance pattern, phage type or plasmid profile among blood culture isolates from infants with bacteremia. Mucocutaneous isolates of C-S from infants with bacteremia were compared with those from infants without invasive disease. Infants with bacteremia had a significantly higher percentage of slime-producing organisms (75% vs. 58%, P = 0.027) and a significantly higher percentage of S. epidermidis species (79% vs. 53%, P = 0.001) than isolates from infants without bacteremia. Our data support the relationship of slime production and the S. epidermidis species of C-S as virulence factors in infants with foreign bodies. Testing C-S for slime production is a relatively simple laboratory procedure which may be an additional aid in the evaluation of their clinical significance.