Frequency, interobserver reproducibility and clinical significance of equivocal peaks in PCR clonality testing using Euroclonality/BIOMED-2 primers

AimsPCR studies for lymphoid clonality are now widely employed, especially using Euroclonality/BIOMED-2 primers. Criteria for interpretation as a clonal result, however, have proven controversial. This study examines the frequency and clinical significance of equivocal amplification patterns and measures the interobserver reproducibility of clonality interpretations.MethodsAt our institution, results of each primer set are first classified as clonal, non-clonal or abnormal (equivocal peak on polyclonal background). Final results for all primer sets are then collectively reported as positive (≥1 clonal result), negative (non-clonal results) or indeterminate (≥1 abnormal result) for a clonal population. Results of 274 consecutive clonality cases were reviewed, and the interobserver reproducibility of individual primer set reactions and final results was determined in a subset of 30 cases.Results44/161 (27%) B-cell and 50/163 (31%) T-cell cases contained at least one abnormal peak. Of these, 29 (64%) and 31 (62%), respectively, showed clonal results in another primer set. Interobserver reproducibility was excellent for most primer sets and for final interpretations, but only fair to good for IGK V-J and TCRB D-J1+2 primer sets. A definitive diagnosis of lymphoma was rendered in 93%, 20% and 6% of B-cell cases and 90%, 42%, and 14% of T-cell cases positive, indeterminate or negative for a clonal population, respectively.ConclusionsUsing a subjective approach, abnormal (equivocal) peaks are frequently observed in routine practice. However, most cases with abnormal peaks contain clonal rearrangements in other primer sets, facilitating overall interpretation of final results with excellent interobserver reproducibility.

Characteristics ofStreptococcus pneumoniaeStrains Colonizing Upper Respiratory Tract of Healthy Preschool Children in Poland

Antibiotic resistant and invasive pneumococci may spread temporally and locally in day care centers (DCCs). We examined 267 children attending four DCCs located in the same city and 70 children staying at home in three seasons (autumn, winter, and spring) to determine prevalence, serotype distribution, antibiotic resistance patterns, and transmission of pneumococcal strains colonizing upper respiratory tract of healthy children without antipneumococcal vaccination. By pheno- and genotyping, we determined clonality of pneumococci, including drug-resistant strains. The average carriage of pneumococci in three seasons was 38.2%. 73.4% and 80.4% of the isolates belonged to serotypes present in 10- and 13-valent conjugate vaccine, respectively. Among the pneumococcal strains, 33.3% were susceptible to all antimicrobial tested and 39.2% had decreased susceptibility to penicillin. Multidrug resistance was common (35.7%); 97.5% of drug-resistant isolates represented serotypes included to 10- and 13-valent conjugate vaccine. According to BOX-PCR, clonality definitely was observed only in case of serotype 14. Multivariate analysis determined DCC attendance as strongly related to pneumococcal colonization in all three seasons, but important seasonal differences were demonstrated. In children attending DCCs, we observed dynamic turnover of pneumococcal strains, especially penicillin nonsusceptible and multidrug resistant, which were mostly distributed among serotypes included to available pneumococcal conjugate vaccines.