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In a prospective observational study, seroconversion to a specific pathogen can serve as a marker of an incident infection, whether or not that infection is symptomatic or clinically diagnosed. While self-reported symptoms can be affected by reporting bias, seroconversion is likely to be free of this bias as it is based on objective measurements of antibody response. Non-invasive salivary antibody tests can be used instead of serum tests to detect seroconversions in prospective studies. In the present study, individuals and families were recruited at a Lake Michigan beach in Wisconsin in August 2011. Data on recreational water exposure and baseline saliva samples (S1) were collected at recruitment. Follow-up data on gastrointestinal symptoms were collected via a telephone interview approximately 10 days post-recruitment. Follow-up saliva samples were self-collected approximately 2 weeks (S2) and 30-40 days post-recruitment (S3) and mailed to the study laboratory. Samples were analyzed for immunoglobulin (Ig) G responses to recombinant antigens of three noroviruses and Cryptosporidium, as well as protein purification tags as internal controls, using an in-house multiplex suspension immunoassay on the Luminex platform. Responses were defined as ratios of antibody reactivities with a target protein and its purification tag. Seroconversions were defined as at least four-fold and three-fold increases in responses in S2 and S3 samples compared to S1, respectively. In addition, an S2 response had to be above the upper 90% one-sided prediction limit of a corresponding spline function of age. Among 872 study participants, there were seven (0.8%) individuals with seroconversions, including six individuals with seroconversions to noroviruses and two to Cryptosporidium (one individual seroconverted to both pathogens). Among 176 (20%) individuals who reported swallowing lake water, there were six (3.4%) seroconversions compared to one (0.14%) seroconversion among the remaining 696 individuals: the crude and age-standardized risk differences per 1000 beachgoers were 32.7 (95% confidence limits 5.7; 59.6) and 94.8 (4.6; 276), respectively. The age-adjusted odds ratio of seroconversion in those who swallowed water vs. all others was 49.5 (4.5; 549), p = 0.001. Individuals with a norovirus seroconversion were more likely to experience vomiting symptoms within 4 days of the index beach visit than non-converters with an odds ratio of 34 (3.4, 350), p = 0.003. This study contributed further evidence that recreational water exposure is associated with symptomatic and asymptomatic waterborne infections, and that salivary antibody assays can be used in epidemiological surveys of norovirus and Cryptosporidium infections., (© 2021. The Author(s).)

Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.

NPM1 gene mutations are the most frequent genetic lesion in the 60% of adult acute myeloid leukemias (AMLs) with normal karyotype and no evidence of typical fusion genes (BCR/ABL1, PML/RARA, AML1/ETO, CBFB/MYH11, DEK/CAN). Using direct sequencing we previously identified six different heterozygous mutants within exon 12 encoding the nucleophosmin C-terminus. Because of these mutations the shuttling protein nucleophosmin is aberrantly delocalized in the cytoplasm of leukemic cells (NPMc+). Here, we designed and tested a denaturing high-performance liquid chromatography (DHPLC) assay to detect NPM1 mutated variants. To assess specificity, sensitivity, reliability, and reproducibility, we analyzed DNA from 120 primary adult AMLs and compared DHPLC results with immunohistochemistry and sequencing. All electropherogram profiles in the 26 NPMc+ leukemias were different from the wild type, indicating 100% sensitivity. Sequencing categorized mutations A, B, and D, and all mutation A cases gave identical elution profiles. The other mutations showed typical chromatograms, with mutations B and D differing for one nucleotide. Elution profiles and sequencing also identified four new variants. Our results suggest that DHPLC detects NPM1mutations as well as direct sequencing and immunohistochemistry, providing a helpful approach in the diagnosis of NPMc+ AML.