Performance of Three Common Hepatitis C Virus (HCV) Genotyping Assays for Identification of HCV Genotype 2/1 Chimeras.

Besides seven major hepatitis C virus (HCV) genotypes (GT), a number of intergenotypic recombinant strains have been described. These so-called chimeras combine genetic characteristics of different HCV genotypes. However, correct genotype classification is important, as choice and duration of direct-acting antiviral (DAA) treatment is mainly based on the viral genotype. Therefore, misclassification of chimeras might lead to suboptimal treatment of patients infected with these strains. For example, 2k/1b chimeras are typically described as HCV genotype 2 strains by commercially available hybridization assays, but real-time PCR-based tests recognizing another HCV region might be more suitable for correct chimera detection. In this study, the analytic capacity of the hybridization-assay Versant HCV Genotype 2.0 (LiPA 2.0) and the real-time PCR-based-assays cobas HCV GT and Abbott RealTime HCV Genotype II were tested in a selected cohort of 230 patients infected with HCV genotype 1 (n = 53) and 2 (n = 177) and 48 patients infected with HCV 2/1 chimeric strains. While the Versant HCV Genotype 2.0 (LiPA 2.0) assay failed to identify chimeras in all of the patients (48/48, 100%), cobas HCV GT and Abbott HCV Genotype II assays identified chimeras correctly in 90% (43/48) and 65% (31/48) of the cases, respectively. In conclusion, while the hybridization-based Versant HCV Genotype 2.0 (LiPA 2.0) assay seems to be unsuitable for detection of HCV 2/1 chimeras, use of the real-time PCR-based assays cobas HCV GT and Abbott RealTime HCV Genotype II led to a higher rate of chimera detection.

A new risk model to predict time to first treatment in chronic lymphocytic leukemia based on heavy chain immunoparesis and summated free light chain.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is frequently accompanied by immune dysregulation. AIMS: In this multicenter prospective study, we investigated whether heavy + light chains (HLC: IgGκ, IgGλ, IgAκ, IgAκ, IgMκ, IgMλ) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) could be used as novel prognostic markers of immunoparesis in 105 treatment-naïve patients with CLL. RESULTS: Heavy + light chains immunoparesis of ≥1, ≥2, and ≥3 isotypes was evident in 74 (70%), 58 (55%), and 36 (34%) patients, respectively. Severe HLC immunoparesis was identified in 40 (38%) patients. Of the IgG subclasses, IgG1 and IgG2 were most frequently suppressed, affecting 46 (44%) and 36 (34%) patients, respectively; 63 (60%) patients had low levels of at least one IgG subclass. In multivariate analysis, severe HLC immunoparesis (hazard ratio [HR]: 36.5; P = .010) and ΣFLC ≥ 70 mg/L (HR: 13.2; P = .004) were the only factors independently associated with time to first treatment (TTFT). A risk model including these variables identified patients with 0, 1, and 2 risk factors and significantly different TTFT (P < .001). Patients with two factors represented an ultra-high-risk group with a median TTFT of only 1.3 months. CONCLUSION: The above findings demonstrate the potential for the use of HLC immunoparesis, together with sFLC measurements, as future prognostic biomarkers in CLL.

Origin, prevalence and response to therapy of hepatitis C virus genotype 2k/1b chimeras.

BACKGROUND & AIMS: Little is known about the epidemiology and frequency of recombinant HCV genotype 2/1 strains, which may represent a challenge for direct antiviral therapy (DAA). This study aims to identify the epidemiology and phylogeny of HCV genotype 2/1 strains and encourages genotype screening, to select the DAA-regimen that achieves the optimal sustained virologic response. METHODS: Consecutive samples from HCV genotype 2 infected patients, according to commercial genotyping, from Germany, Italy and Israel were re-genotyped by Sanger-based sequencing. Virologic, epidemiological, and phylogenetic analyses including other published chimeras were performed. RESULTS: Sequence analysis of 442 supposed HCV genotype 2 isolates revealed 61 (genotype 2k/1b (n=59), 2a/1b (n=1) or 2b/1a (n=1)) chimeras. No chimeras were observed in Italy, but the frequency was 14% and 25% in Germany and Israel. Treatment of viral chimera with sofosbuvir/ribavirin led to virologic relapse in 25/27 patients (93%). Nearly all patients treated with genotype 1-based DAA-regimens initially (n=8/9), or after relapse (n=13/13), achieved a sustained virologic response. Most patients with 2k/1b chimeras (88%) were originally from eight different areas of the former Soviet Union. All known 2k/1b chimeras harbour the same recombination breakpoint and build one phylogenetic cluster, while all other chimeras have different phylogenies. CONCLUSIONS: The HCV genotype 2k/1b variant derives from one single recombination event most likely in the former Soviet Union, while other chimeras are unique and develop independently. A relatively high frequency has been observed along the migration flows, in Germany and Israel. In countries with little migration from the former Soviet Union the prevalence of 2k/1b chimeras is expected to be low. Treatment with sofosbuvir plus ribavirin is insufficient, but genotype 1-based regimens seem to be effective. Lay summary: The frequency of recombinant HCV is higher than expected. A novel recombinant variant (HCV genotype 2a/1b) was identified. Screening for recombinant viruses would contribute to increased response rates to direct antiviral therapy.

Auto-validation of complete blood counts in an outpatient's regional laboratory.

BACKGROUND: The rate of auto-validation is dependent on the ability of the laboratory information system (LIS) to integrate historical data, on the frequency and methods for identifying analyzer errors, and on the criteria for reflex testing, including the need for peripheral smear review. The rate of auto-validation in outpatient laboratories, however, is unclear. METHODS: We examined 45,925 consecutive complete blood count (CBC) test results (1 January, 2014-31 January, 2014) from patients aged 50±24 years. The LIS auto-validates all samples according to set criteria. Technicians validated test results when previous CBC test results were required to determine: 1) the need for peripheral slide review and/or sample rerun or 2) the need for reflex testing to detect autoimmune hemolytic anemia or ß-thalassemia minor. RESULTS: The auto-validation rates were 97.6% after rejecting results requiring validation to determine the need for a peripheral smear review and/or sample rerun. This decreased to 92.9% after including reflex testing to determine the reasons for normocytic and microcytic anemia. We estimated that auto-validation decreased the workload by 7.7-11.6 h per 3000 test results. CONCLUSIONS: We conclude that very high auto-validation rates are possible in outpatient general laboratories, leading to conformity in the validation process and a considerable estimated savings in technician time. Further studies are needed in other settings.

Triple positive antiphospholipid antibody profile in outpatients with tests for lupus anticoagulants.

BACKGROUND: A triple positive antiphospholipid (aPL) antibody profile [two positive serum IgG aPL antibodies along with one positive functional plasma lupus anticoagulant (LAC) test result] is associated with an increased risk for thrombosis, whereas patients with single positive test results may have little to no increased risk. The frequency of triple positivity in outpatients with various combinations of LAC test results is unclear. METHODS: We extracted from our database all LAC test results [dilute Russell viper venom times (dRVVT) and silica clotting times (SCT)] that had concomitant serum IgG aPL testing [both serum anti ß2-glycoprotein I (anti-ß2GPI) and anti-cardiolipin (aCL) antibodies]. RESULTS: There were 3195 patients without a prolonged prothrombin time. Double antibody positivity was found in 1% (31/2955) of those with normal functional LAC test results, in 16.0% (31/81) of those with a positive dRVVT, in 12.7% (10/79) of those with a positive SCT, and in 56.3% (45/80) of those with both tests positive (p<0.001). A triple positive aPL antibody profile was found in 28.3% (68/240) of those with at least one positive LAC test result. CONCLUSIONS: We conclude that 28% of patients with elevated LAC tests have a triple positive aPL antibody profile and patients with two positive LAC tests have a higher prevalence of a triple positive profile than do those with one positive LAC test result.

Association of Vitamin B12 Deficiency with Homozygosity of the TT MTHFR C677T Genotype, Hyperhomocysteinemia, and Endothelial Cell Dysfunction.

BACKGROUND: Hyperhomocysteinemia is associated with increased cardiovascular risk, but treatment with folic acid has no effect on outcome in unselected patient populations. OBJECTIVES: To confirm previous observations on the association of homozygosity for the TT MTHFR genotype with B12 deficiency and endothelial dysfunction, and to investigate whether patients with B12 deficiency should be tested for 677MTHFR genotype. METHODS: We enrolled 100 individuals with B12 deficiency, tested them for the MTHFR C677T polymorphism and measured their homocysteine levels. Forearm endothelial function was checked in 23 B12-deficient individuals (13 with TT MTHFR genotype and 10 with CT or CC genotypes). Flow-mediated dilatation (FMD) was tested after short-term treatment with B12 and folic acid in 12 TT MTHFR homozygotes. RESULTS: Frequency of the TT MTHFR genotype was 28/100 (28%), compared with 47/313 (15%) in a previously published cohort of individuals with normal B12 levels (P = 0.005). Mean homocysteine level was 21.2 ± 16 µM among TT homozygotes as compared to 12.3 ± 5.6 µM in individuals with the CC or CT genotype (P = 0.008). FMD was abnormal ( 6%) in 9/13 TT individuals with B12 deficiency (69%), and was still abnormal in 7/12 of those tested 6 weeks after B12 and folic treatment (58%). CONCLUSIONS: Among individuals with B12 deficiency, the frequency of the TT MTHFR genotype was particularly high. The TT polymorphism was associated with endothelial dysfunction even after 6 weeks of treatment with B12 and folic acid. Based on our findings we suggest that B12 deficiency be tested for MTHFR polymorphism in order to identify potential vascular abnormalities and increased cardiovascular risk.

Criteria for reflex peripheral smear review in infants.

Criteria for peripheral smear review are designed to include those samples with results outside the reference interval and can be more extreme based on what is considered to have clinical utility. However, we are unaware of previous studies that reported the distributions of various complete blood cell count (CBC) parameters in infants. In the following study we reviewed screening CBC results of 692 infants aged 9-15 months in order to determine the proportion of peripheral smear reviews recommended according to consensus criteria and that after adjusting for the observed distributions of the various parameters. According to consensus criteria the recommended reflex peripheral smear review rate was 39.7% (95% CI 36.1-43.4) whereas after adjustment for the observed distributions, the rate fell to 5.6% (95% CI 3.9-7.3) (p < 0.001). The major reasons for the difference in rates were the high proportion of infants with an absolute lymphocyte count > 7 × 10(9)/L (17.5%), the presence of a plus one blast flag (4.3%), and a large unstained cell count of ≥ 5% (26.2%) (equivalent to + 1 atypical flag). We found that international consensus criteria for reflex peripheral smear review results in a very high peripheral smear review rate in well infants, and might be inappropriate.

The influence of vitamin D supplementation on melatonin status in patients with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) incidence is higher in geographic regions with less sunlight exposure. Both vitamin D and melatonin are essential mediators of the effect of sunlight in health, and as such are candidates to play a key role in MS. We hypothesized that vitamin D and melatonin may have related influences in patients with MS. METHODS: In a randomized, double blind study of 40 IFN-ß treated MS patients, 21 patients were assigned to 800 IU of vitamin D3 per day (low dose), while 19 patients received 4,370 IU vitamin D3 per day (high dose) for one year. Serum 25-hydroxy-vitamin-D (25-OH-D) and nighttime urine melatonin metabolite, 6-sulphatoxy-melatonin (6-SMT), were measured at baseline, 3 months and 1 year from enrolment. RESULTS: After 3 months supplementation, 25-OH-D levels increased and nighttime melatonin secretion decreased significantly in the high dose group, but not in the low dose group. After 1 year, a decrease in 25-OH-D levels, accompanied by an increase of urine nighttime 6-SMT were observed in the high dose group. Percent change in serum 25-OH-D was significantly and negatively correlated with percent change in urine 6-SMT after 3 months and between 3 months to 1 year. 25-OH-D levels by the end of the study were significantly and negatively correlated to BMI. CONCLUSIONS: Melatonin secretion is negatively correlated with alterations in serum 25-OH-D in IFN-ß treated patients with MS. The finding suggests that melatonin should be considered as a potential mediator of vitamin D neuro-immunomodulatory effects in patients with MS.

Vitamin D supplementation for patients with multiple sclerosis treated with interferon-beta: a randomized controlled trial assessing the effect on flu-like symptoms and immunomodulatory properties.

BACKGROUND: Flu-like symptoms (FLS) are common side effects of interferon beta (IFN-ß) treatment in patients with Multiple Sclerosis (PwMS) and are associated with post-injection cytokine surge. We hypothesized that vitamin D3 supplementation would ameliorate FLS by decreasing related serum cytokines' levels. METHODS: In a randomized, double blind study of 45 IFNß-treated PwMS, 21 patients were assigned to 800 IU of vitamin D3 per day (low dose), while 24 patients received 4,370 IU per day (high dose) for one year. FLS were assessed monthly by telephonic interviews. Serum levels of 25-hydroxy-D (25-OH-D), calcium, PTH, IL-17, IL-10 and IFN-γ were measured periodically. EDSS, relapses, adverse events and quality of life (QoL) were documented. RESULTS: 25-OH-D levels increased to a significantly higher levels and PTH levels decreased in the high dose group. There was no significant change in FLS. IL-17 levels were significantly increased in the low dose group, while patients receiving high dose vitamin D had a heterogeneous IL-17 response. No significant differences in relapse rate, EDSS, QoL, serum IL-10 and IFNγ were found. Hypercalcemia or other potential major adverse events were not observed. CONCLUSION: Vitamin D supplementation to IFN-ß treated PwMS, at the doses used, seems safe and associated with dose-dependent changes in IL-17 serum levels, while not affecting IFN-ß related FLS. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT01005095.

Testing for lupus anticoagulants--fresh or frozen?

BACKGROUND: There are no previous studies reporting the effect of using frozen-thawed plasma on lupus anticoagulant ratios in kits with the combined screen and confirm assay. METHODS: In the following study we chose patients with elevated dilute Russel's viper venom test (dRVVT) normalized ratios and compared the test results of fresh to frozen-thawed plasma. Platelet counts ranged from 2 to 7×10(3)/µL (10(9)/L) after a second centrifugation before freezing. RESULTS: There were 13 out of 14 dRVVT test normalized ratios that decreased after freezing (p<0.001), leading to the misclassification of six of 14 patients with high values that decreased into the reference interval. CONCLUSION: The major finding of this study is that testing frozen-thawed plasma with platelet counts <10,000/µL (10(9)/L) results in a significant decrease in dRVVT ratios. Although there was a consistent decrease in SCT normalized ratios as well, it did not lead to misclassifications.

The Concordance between Mumps and Rubella Sero-Positivity among the Israeli Population in 2015.

Mumps and rubella are vaccine-preventable viral diseases through the measles-mumps-rubella-varicella (MMRV) vaccine, administered at 12 months and again at 6 years. We assessed the sero-prevalence of mumps and rubella, identified factors associated with sero-negativity, and evaluated concordance between mumps and rubella sero-positivity. A national cross-sectional sero-survey was conducted on samples collected in 2015 by the Israel National Sera Bank. Samples were tested for mumps and rubella IgG antibodies using an enzyme-linked immunosorbent assay. Of 3131 samples tested for mumps IgG, 84.8% (95%CI: 83.5-86.0%) were sero-positive. Sero-negativity for mumps was significantly associated with age (high odds ratios observed in infants younger than 4 years and 20-29 years old subjects). Of 3169 samples tested for rubella IgG antibodies, 95.2% (95%CI: 94.4-95.9%) were sero-positive. Rubella sero-negativity was significantly associated with age (high odds ratios observed in children younger than 4 years old and adults older than 30 years), males, Jews, and others. Concordant sero-positivity for both mumps and rubella viruses was observed in 83.9% of the tested samples. The Israeli population was sufficiently protected against rubella but not against mumps. Since both components are administered in the MMRV vaccine simultaneously, the mumps component has a lower uptake than rubella and quicker waning.

Prevalence and course of pseudothrombocytopenia in outpatients.

BACKGROUND: The prevalence and course of pseudothrombocytopenia in outpatients is uncertain. METHODS: In a cohort study of 687,955 members of a health maintenance organization, we extracted 36,780 consecutive automated complete blood count test results and determined the point prevalence of pseudothrombocytopenia during a one-month period. We also calculated a retrospective cumulative prevalence over the past 5 years. RESULTS: There were 1105 (2.7%) patients with platelet counts of 100-149×10(9)/L and 304 (0.8%) with counts <100×10(9)/L, of whom 12.8% (n=39) had pseudothrombocytopenia newly discovered or in the past. There were 40 additional patients with a history of pseudothrombocytopenia, but now showing platelet counts of 150×10(9)/L or more, and another 21 with platelet counts of 100-149×10(9)/L. The total cumulative prevalence of pseudothrombocytopenia was 0.27% (100/36,780), 2.5-fold higher than the point prevalence of 0.11% (39/36,780). Platelet counts were extremely variable in these patients during the 5-year follow-up period. CONCLUSIONS: Physicians should be aware of the variability of platelet counts in patients with pseudothrombocytopenia in order to reduce unnecessary retesting. Since pseudothrombocytopenia can result in platelet counts between 100 and 149×10(9)/L, peripheral smears should be done in all patients with such values, or the reference limit value should be lowered to 100×10(9)/L.

Lupus anticoagulant testing: analyzing fresh samples after a single centrifugation and after a 6-8 h delay.

BACKGROUND: For automation it is important to know the effect of analyzing fresh samples after a single rather than after two centrifugations, and to determine test stability. METHODS: This study compared silica clotting times (SCT) and dilute Russel's viper venom tests (dRVVT) after one and two centrifugations in 50 fresh plasma samples. Then it compared test results within 4 h to those after 6-8 h of blood drawing in 40 samples. Means, minimums, maximums and quartiles of the paired screen were compared, test ratios were confirmed and correlations, linear regressions and Bland-Altman statistics were calculated. RESULTS: The distributions of test results were nearly identical, regardless of the number of centrifugations or timing of the analysis. The first centrifugation explained 97.7% and 94.8% of the variance of tests results after the second centrifugation for the SCT and dRVVT, respectively. The test results after 6-8 h explained 98.3% and 96.3% of the variance of the SCT-ratios and dRVVT-ratios, respectively, tested within 4 h. Inter-day coefficients of correlation of ratio comparisons were similar to those of the controls values. CONCLUSIONS: Testing of fresh samples after a single centrifugation might replace batch testing of frozen samples after double centrifugation, providing timelier reporting of results and resulting in savings of technician time.

Seropositivity of measles antibodies in the Israeli population prior to the nationwide 2018 - 2019 outbreak.

Measles vaccine is administered in Israel as part of the routine childhood immunization program, at ages 1 and 6 years. In this study, we assessed seropositivity of the Israeli population against measles before the onset and propagation of the 2018-2019 measles outbreak. From the Israel Center for Disease Control National Serum Bank, 3,164 samples collected during 2015 were tested for measles antibodies. All the tests were performed using Enzyme-Linked Immunosorbent Assay (ELISA) commercial kit (Enzygnost, Anti-Measles Virus/IgG: Behring, Marburg, Germany). The overall seropositivity rate for measles was 90.7%. The seropositivity rate at 6 months and younger was 48.9%, and decreased to 3.8% among infants aged 6-11 months. Seropositivity increased to 90.7% in the 1-4-year age group, and reached 96.1% for 5-9 year-old children. Our results suggest high immunity in the Israeli population against measles virus, but not high enough to prevent outbreaks because of pockets of specific population groups with low immunization coverage. Infants between ages 6 and 11 months and children younger than 2 years had the lowest seropositivity rates being the age groups with the highest attack rates of measles during the epidemic of 2018. Efforts should be aimed at avoiding any delay in vaccination once a child reaches the age of 1 year and improving immunity levels in children aged 1-4 years.

Is the association between Helicobacter pylori infection and anemia age dependent?

BACKGROUND: The relationship between H. pylori infection and anemia in childhood is still unclear. The aim of the study was to examine the association between H. pylori infection and anemia or iron deficiency in school-age children and in infants. MATERIALS AND METHODS: Six- to 9-year-old Israeli Arab children (N = 202) and infants (N = 197) were examined for hemoglobin and ferritin levels. ELISA was used to detect H. pylori antigens in stool specimens collected from the participants. Household characteristics were obtained through personal interviews with the mothers. RESULTS: The prevalence of anemia was 15.5 versus 5.5% in H. pylori-positive and -negative school-age children, respectively and 34.5 versus 29.8% in H. pylori-positive and -negative infants, respectively. The Mantel-Haenszel age-adjusted prevalence ratio (PR) and 95% confidence intervals (CIs) were 1.6 (95%CI 1.0, 2.6). In multivariate analysis controlling for socioeconomic variables, H. pylori infection was associated with 2.8 higher prevalence of anemia only in school-age children: adjusted PR 2.8 (95% CI 0.9, 9.3). The adjusted mean difference in hemoglobin levels between H. pylori infected school-age children and uninfected ones was -0.372 gr/dL (95% CI -0.704, -0.039) (p = .04). The respective mean ferritin difference was -6.74 µg/L (95% CI -13.38, -.011) (p = .04). Such differences were not found in infants. CONCLUSIONS: H. pylori infection is associated with higher prevalence of anemia in school-age children independently of socioeconomic variables. Such association was not observed in infants. These findings are of clinical and public health importance.

Microscopic or occult hematuria, when reflex testing is not good laboratory practice.

Consensus opinion suggests that hematuria found by dipstick and not confirmed on microscopic examination (<2 erythrocytes per high power field) signifies a false-positive reagent strip test result. Standard practice is to repeat the dipstick test several days later and if still positive to confirm by microscopic examination. If discordant results are obtained, experts recommend reflex testing for urinary myoglobin and hemoglobin concentrations. The question is whether or not this approach represents good laboratory practice. These recommendations are not evidence based. We conclude that the reference range for red blood cells on the reagent strip should be increased to 25x10(6) cells/L for young men, and 50x10(6) cells/L for the rest of the adult population, ranges consistent with flow cytometry reports. Confirmation reflex testing using tests that have inferior sensitivity, precision and probably accuracy is not recommended.

The rate of manual peripheral blood smear reviews in outpatients.

BACKGROUND: The International Consensus Group for Hematology Review recommends manual blood smears for new findings from automated complete blood cell (CBC) tests. However, in outpatients, the rate of peripheral manual blood smear reviews is uncertain, as is the effect of historical results on this rate. METHODS: In a prospective cohort study of 687,955 members of a health maintenance organization, we extracted 39,759 consecutive automated CBC test results and determined the rate of manual peripheral smear reviews that were indicated using different triggering criteria, and historical results. RESULTS: Individual triggering values increased from 10.7% to 19.5% when comparing our criteria to those of the consensus criteria. The proportion of CBC test results with at least one triggering value was 7.2% using our criteria, compared to 13.9% with the inclusion of a mean corpuscular volume <75 and a plus one immature granulocyte flag according to the consensus committee. Availability of historical data decreased our peripheral smear review rate to 2.6%. CONCLUSIONS: The peripheral smear review rate in outpatients using the consensus criteria was higher than with our less stringent criteria. If historical data are available and no clinical utility is demonstrated for the additional consensus criteria, an acceptable out-patient review rate might be as low as 2.6%.