Determinants of Protection Against Measles Infection in a Vaccinated Healthcare Worker

Background: In 2019, a measles community outbreak resulted in a secondary case in a health care worker (HCW) working in a pediatric hospital in Montral, Canada. Following the event, HCWs were screened to identify individuals susceptible to measles infection based on serology results. Objective: Our aim was to assess measles seroprotection rates and to evaluate vaccine responses of susceptible HCWs using commercial enzyme immunoassay (EIA) or enzyme linked immunosorbent assay (ELISA). Methods: Emergency department (ED) employees, including doctors, were screened for measles susceptibility as part of a postoutbreak measure by the hospital occupational health service. Demographic information was collected. Measles history and vaccination information were collected using a personal vaccination booklet, employee vaccination profile, or the Qubec vaccination registry. According to the Quebec Immunization Protocol (PIQ), individuals born before 1970, or who have received 2 doses of a measles-containing vaccines are considered protected. Individuals with undetectable or equivocal antibody levels were considered at risk of measles infection. These individuals were offered vaccination and were tested for vaccine response 4 weeks after vaccination. Results: Anti-IgG measles antibody results, demographic information, and vaccination information were obtained for 257 employees. The results are currently available for 233 HCWs: 224 HCWs (96%) were seropositive, 7 (3%) were seronegative, and 2 were equivocal. Among seronegative individuals, 6 (85.7%) were born after 1980 and 3 (42.9%) had received 2 doses of a measles-containing vaccine. Of those with an equivocal result, 1 (50%) had received 2 doses and 1 (50%), born after 1970, did not confirm vaccination status. Finally, 9 (4%) of seropositive individuals were not vaccinated; of whom 8 (88.9%) were born before 1970. Conclusions: Our preliminary results suggest that the 95% immunity threshold that is usually required to prevent secondary transmission of measles has been reached in our ED HCW cohort. Even years after the second MMR dose, HCWs remain well protected. Relying on documented vaccination status is thus acceptable.Funding: NoneDisclosures: None

Differential Diagnosis of Multiple Primary and Intra-lung Metastasis of Lung Cancer by Multiple Gene Detection

Objective To study the differential diagnosis of MPLC and IM by detecting the different lesions of the same patient. To explore the differences in prognosis between MPLC and IM, and to explore the factors affecting the prognosis of multi-focal lung cancer. Methods Fifty patients with multi-focal lung cancer were screened, and the relevant clinical information was noted; the patients were diagnosed by ACCP standard. Mutations of the lesions were detected by ARMS-PCR, and the detected genes included EGFR, ALK, ROS1, MET, KRAS, RET, HER-2, BRAF, NRAS and PIK3CA. The results of genetic testing were compared with those of ACCP standard diagnosis. Results We analyzed a total of 101 tumors from 50 patients. Classification based on gene testing contradicted the clinicopathologic diagnosis in 10 (20%) of the comparisons, identifying independent primaries in 6 cases diagnosed as metastasis and metastases in 4 cases diagnosed as independent primaries. Another 7(14%) tumor pairings were assigned an “equivocal” result based on gene testing. The results of gene testing of the remaining 33(66%) tumor pairings were consistent with the clinicopathologic diagnosis. The mutant heat map indicated that IM patients have a higher rate of mutation consistency than MPLC patients. Conclusion Multi-gene detection of multi-focal lung cancer has a certain auxiliary effect on the differential diagnosis of MPLC and IM, which can complement the clinical standards, but also has some limitations.

Analysis of Correlation and Agreement between the Uroflowmetry and the International Prostate Symptom Score in Patients after retropubic Radical Prostatectomy: A Multicenter Prospective Study

Objectives Patients undergoing retropubic radical prostatectomy (RRP) may suffer from lower urinary tract symptoms (LUTS). We aim to characterize LUTS and to evaluate the correlation and agreement between uroflowmetry and the International Prostate Symptom Score (IPSS) in patients after RRP in two reference centers. Methods An observational multicenter prospective study was conducted between December 2015 and September 2016. Patients with at least 12-months of follow-up after RRP were included; these were evaluated with uroflowmetry and the IPSS. Results A total of 90 patients were included. The mean follow-up was of 54.6 months (standard deviation [SD] = 27.52), and the mean age was 65 (SD = 6.85) years old. The mean IPSS was 7.41 (SD = 6.29), with 33.3% (n = 54) of the patients with moderate symptoms and 6.7% (n = 6) with severe symptoms. A total of 50% (n = 45) of the patients had normal uroflowmetry. Patients with an abnormal/equivocal result in the uroflowmetry had a mean of 9.31 (SD = 7.03) points in the IPSS versus 5.51 (SD = 4.82) in patients with a normal uroflowmetry result (p < 0.01). The level of agreement between mild versus moderate-to-severe LUTS and normal uroflowmetry versus abnormal/equivocal was 61.1% (k = 0.22, p = 0.04). We found that a score ≥ 10 in the IPSS had a level of agreement of 65.6% (k = 0.31, p = 0.0004). Conclusions We consider that although the IPSS cannot replace uroflowmetry and vice versa, these tests are complementary and may be useful tools in the evaluation of patients with LUTS after RRP.

Correlation of Flow Cytometric Aberrations with Cytogenetic, Molecular Genetic, and Morphology in Patients with Unexplained Cytopenias

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had >1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

TOXOPLASMOSIS: SEROLOGIC PROFILE OF PREGNANT WOMEN AT THE BRAZZAVILLE UNIVERSITY CENTRE HOSPITAL (CHUB)

Introduction: Toxoplasmosis is a disease caused by an obligate intracellular coccidia Toxoplasma gondii, which is transmitted by cats. In pregnant women, it is a concern because of the severe complications to the foetus. The objective of this study is to determine the toxoplasma serologic profile in pregnant women at the Brazzaville University Centre Hospital (CHUB). Materials and Methods: This is a cross-sectional study from September 2015 to March 2016 (6 months) which includes sera from pregnant women received at the Parasitology Mycology laboratory of the CHUB for Toxoplasma serology. Immunoglobulins G and M searches were done by immuno-analysis (Biomerieux, Mini-Vidas technology). The data was analysed by the IBM SPSS version 20 software. The comparisons of proportion is done by the khi 2 test. The level of significance of statistical data were fixed at 5%. Results: The mean age of pregnant women included in our study was 27, 8+/- 6,84 with the extremes ages of 15 and 44 years. Toxoplasma seroprevalence in this study is 47,2% (68/144). The types of immunoglobulins (Ig) retrieved were IgG alone in 45,1% of cases (65/144), IgG associated to IgM in 2,8% of cases (4/144). Serological profiles were: no immunity (52,1%) immunised (41%), recent infection (1,4%) active infection (2,8%) equivocal result (2,8%). Conclusion: Toxoplasmosis is a zoonosis which represents a real public health issue in our environment, even when the level of immunised pregnant women seems high.

Screening for Lynch Syndrome by Immunohistochemistry of Mismatch Repair Proteins: Significance of Indeterminate Result and Correlation With Mutational Studies

Context.— Immunohistochemical expression of mismatch repair (MMR) protein is a well-accepted method for routine screening for Lynch syndrome with relatively high sensitivity and specificity. Occasionally, however, immunohistochemistry (IHC) can yield an equivocal result with poor reproducibility and the potential for misdiagnosis. Objective.— To determine the frequency and significance of indeterminate MMR IHC expression in patients routinely screened for Lynch syndrome and correlation with germline mutation studies. Design.— Semiquantitative scoring of MMR IHC was performed by image analysis in 479 cases, of which 380 were colorectal and 99 endometrial cancer. Scores of 10% or more, less than 10%, and 0% were used as cutoffs for retained, indeterminate, and loss of expression, respectively. Negative and indeterminate IHC results were confirmed by mutational studies. Results.— Four hundred eighteen of 479 cases (87.2%) were reported as retained expression, 45 (9.3%) as loss of expression, and 16 (3.3%) as indeterminate expression. Fifteen of 45 (33.3%) and 8 of 16 (50%) with loss and indeterminate expression, respectively, were found to have Lynch syndrome by germline studies. The overall frequency of Lynch syndrome in our patient population was 4.8% (23 of 479), and 34.7% of these (8 of 23) were associated with indeterminate IHC expression. In the indeterminate group, MLH1 germline mutation was the most frequent (6 of 13; 46.2%), followed by MSH6 (4 of 13; 30.7%). Conclusions.— Our findings provide further evidence that indeterminate IHC should be further investigated for possible MMR germline mutation. Guidelines for interpretation of MMR IHC and the establishment of more objective criteria for defining indeterminate results are important to improve the sensitivity and specificity of the IHC assay.

The use of local isolates in Western blots improves serological diagnosis of Lyme disease in Scotland

Nine Scottish Borrelia burgdorferi isolates were investigated in IgG Western blot tests. Sera previously found to be positive and negative when tested by routine Western blots prepared from reference strain B. burgdorferi sensu stricto antigen had different outcomes with these isolates. Two isolates, E5 (Borrelia afzelii) and G4 (B. burgdorferi sensu stricto) performed well, reproducing Western blot-positive results in 90 and 95 % of tests, respectively. When antigens from both isolates were incorporated into a single IgG Western blot, the results of a panel of sera were improved when compared to the routine reference strain IgG Western blot. All of the sera positive by the routine Western blot remained positive using the Scottish isolate antigen mix. Twenty-three of the 25 negative sera remained negative and two produced an equivocal result. Of the 15 samples that tested IgG Western blot equivocal with the B. burgdorferi sensu stricto reference strain, 11 (73 %) became weak or strong positive when tested with the B. afzelii/B. burgdorferi sensu stricto antigen mix (χ 2=14.35, Yates' correction, P<0.001). In seven of these, a clinical picture of Lyme disease was consistent with the new results. The use of Scottish strains of B. afzelii and B. burgdorferi sensu stricto to provide antigen for the IgG Western blot improves the diagnosis of Lyme disease for patients in Scotland.

Zap70 Methylation Correlates with Zap70 Expression in Chronic Lymphocytic Leukemia.

ZAP70 expression in chronic lymphocytic leukemia (CLL) correlates with the presence of unmutated immunoglobulin chain (IgVH) genes and with poor clinical outcome. We hypothesised that expression of ZAP70 may be related to the methylation status of the ZAP70 gene. Bisulphite sequencing of a 5′ region of the ZAP70 gene showed a significant difference in CpG methylation between ZAP70 positive and ZAP70 negative cells. We developed a bisulfite restriction PCR assay, insensitive to 10% T cell contamination, to determine the methylation status of a CpG dinucleotide 332 base pairs downstream of the transcription start site, which represented methylation across the sequenced region. This assay was used to detect ZAP70 methylation in 87 patients with CLL, 12 patients with mantle cell lymphoma (MCL) and 12 patients with splenic marginal zone lymphoma (SMZL). ZAP70 protein expression and IgVH gene mutation status had been previously established in all cases. Global cytosine methylation was also analysed in a subset of 15 of the CLL patients by the capillary electrophoresis with laser-induced fluorescence method. Considering the CLL cases, the ZAP70 gene was methylated in 53 patients, unmethylated in 32 patients and an equivocal result was obtained for 2 patients. Correlating methylation status with ZAP70 expression, 51 of 53 (96%) ZAP70 positive patients were methylated and 30 of 32 (94%) ZAP70 negative patients were unmethylated (p<0.0001). Similarly, 49 of 54 (91%) patients with mutated IgVH genes were methylated and 27 of 31 (87%) unmutated patients were unmethylated (p<0.001). The median survivals of methylated and unmethylated CLL patients were 211 and 85 months respectively (p<0.0001) which was very similar to that found for ZAP70 expression or IgVH gene mutational status. The mean global cytosine methylation level was 3.9±0.15% in the ZAP70 methylated CLL patients and 4.1±0.12% in the ZAP70 unmethylated CLL patients; the difference between the two patient groups was significant (p=<0.001) which implies that if ZAP70 methylation is malignancy-related, then hypomethylation of ZAP70 in a subset of CLL arises as a consequence of gene specific rather than global hypomethylation. Among the 24 MCL and SMZL patients an absolute correlation was seen between ZAP70 expression and methylation. However, in marked contrast to the correlation seen in CLL, all 7 non-CLL patients with unmutated IgVH-genes and 16 of 17 non-CLL patients with mutated IgVH-genes showed methylated ZAP70. Therefore, measurement of ZAP70 gene methylation is a simple and reproducible method for predicting prognosis in CLL, which correlates closely with ZAP70 expression and IgVH gene mutational status. The assay is suitable for stored DNA and histological samples as well as for fresh or DMSO-frozen cells. In addition, the methodology of our assay presents none of the standardisation problems, which are relevant to the current flow cytometric assays for ZAP70.

Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioMérieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.