No abstract

SUMMARYClonal changes of serotype 19A pneumococci have been appreciated in conjunction with growing prevalence of this serotype after implementation of the seven-valent pneumococcal conjugate vaccine (PCV7). In the present study, we characterized serotype 19A pneumococci collected in Russia within a decade preceding the implementation of PCV vaccination and described their clonal evolution. We retrospectively analyzed non-invasive serotype 19A isolates collected in 2002-2013. All isolates were subjected to multilocus sequence typing, antimicrobial susceptibility testing, determination of macrolide resistance genotype, molecular detection of pilus islet (PI) carriage, sequencing of penicillin-binding protein (PBP) genes. A total of 49 serotype 19A isolates represented 25 sequence types, of which 14 were newly described. The majority of isolates were distributed among clonal complex (CC) 663 (28%), CC230 (25%), CC156, and CC320 (14% each). CC663 and CC156 dominated in 2003, but were replaced by CC230 and CC320 later on; CC320 was only evident starting 2010. All isolates of CC663 and CC156 carried PI1; CC320 possessed both PI1 and PI2. The overall rate of altered amino acids in penicillin-nonsusceptible isolates was 13·9%, 7·2%, and 8·7% for PBP1a, PBP2b, and PBP2x, respectively. Our findings demonstrate that the clonal structure of serotype 19A pneumococci may evolve without PCV pressure.

Reverse-transcription PCR (RT-PCR) is considered the most sensitive method for the detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, this method is relatively resource-and time-consuming. This study was performed to compare SARS-CoV-2 nucleocapsid antigen (N-Ag) testing using an enzyme-linked immunosorbent assay (ELISA) with SARS-CoV-2 RNA detection. Methods: Parallel SARS-CoV-2 RT-PCR and quantitative N-Ag ELISA analysis was executed on nasopharyngeal specimens obtained during SARS-CoV-2 screening in a cohort of pre-hospitalization patients. Results: In total, 277 specimens were examined, including 182 (65.7%) RT-PCR-positive specimens, which demonstrated a median cycle threshold (Ct) value of 27 (interquartile range (IQR) 23-35). The SARS-CoV-2 N-Ag was detected in 164 of the 182 RT-PCR-positive specimens (overall sensitivity 90.1%). Among the 95 RT-PCR-negative specimens, 72 were N-Ag-negative (specificity 75.8%). SARS-CoV-2 RT-PCR and N-Ag ELISA results demonstrated a strong agreement (Cramer's V = 0.668; P < 0.001). N-Ag concentrations spanned from 5.4 to 296 000 pg/ml (median 901 pg/ml, IQR 43-1407 pg/ml) and were inversely correlated with Ct values (Spearman's r = À0.720; P < 0.001). Conclusions: SARS-CoV-2 N-Ag ELISA results were in close agreement with RT-PCR results, and N-Ag concentrations were proportional to viral loads. Thus, SARS-CoV-2 quantitative antigen testing could be an additional diagnostic instrument for SARS-CoV-2.

Laboratory diagnostics of lysosomal acid lipase deficiency (LAL‐D), a rare disorder associated with LIPA alterations, are based on the evaluation of LAL activity. In dry blood spots (DBS) submitted for LAL‐D diagnostics (the screening cohort) over a two‐year period or obtained from a cohort of retrospective LAL‐D patients, we measured: (1) LAL activity using a two‐reaction assay with 4‐methylumbelliferone palmitate (4‐MU‐Palm) and Lalistat‐2, a specific LAL inactivator; (2) total lipase (TL) activity by a 1‐hour kinetic 4‐MU‐Palm cleavage reaction (no Lalistat‐2). The TL activity was expressed as the area under the kinetic curve after 1 hour (TL‐AUC 1h ) of the reaction and presented as the median (min‐max). LAL activity was reduced in 30/537 individuals from the screening cohort, among which LIPA sequencing revealed six patients and one carrier. Overall, 16 (89%) individuals among six novel and 12 retrospective LAL‐D patients carried at least one c.894G>A mutation (six were homozygous). The TL‐AUC1h in nonLAL‐D specimens with normal LAL activity (n = 90) was unambiguously higher (9471 [4015‐23 585] RFU*h/punch) compared to LAL‐D patients, including six new and nine retrospective patients (1810 [357‐2608] RFU*h/punch). Importantly, in 13/15 examined nonLAL‐D specimens with reduced LAL activity the TL‐AUC1h was above a threshold of 2652 RFU*h/punch. Applying this threshold, the TL‐AUC1h index discriminated all LAL‐D patients (100% sensitivity) and 103/105 nonLAL‐D specimens (98% specificity). Given that there is no need for Lalistat‐2 and two parallel enzymatic reactions in conjunction with high sensitivity and specificity, the kinetic assay seems to be practical for LAL‐D screening. Synopsis Lysosomal acid lipase deficiency responsible for Wolman disease and cholesterol ester storage disease could be reliably detected using a kinetic assay of total lipase activity with a fluorogenic substrate.

No abstract