Prospective validation of an automatic reflex test for identifying spurious elevations of mean corpuscular haemoglobin concentration due to the presence of cold agglutinins.

Cold agglutinins (CA) in blood may cause false reduction in red blood cell (RBC) count and false increases of RBC indices, such as mean corpuscular haemoglobin concentration (MCHC). Preheating at 37 °C for 2 h is used to overcome this problem. We previously proposed the integration in a total laboratory automation (TLA) setting of a customized reflex test in the presence of MCHC >385 g/L for identifying spurious elevations due to CA. Here, we prospectively evaluate this approach after its introduction in our clinical practice. We evaluated 73 consecutive blood samples from 34 adult patients. Short heating (<1 min) at 41 °C using the reticulocyte channel of Sysmex XN-9000 platform was followed by calculation of optical parameters by the instrument software to ensure quick solution of the CA-dependent problems. After the reflex test in the reticulocyte channel, MCHC dropped below 385 g/L in 50 samples. The reflex markedly corrected the RBC number in eight samples obtained from three patients with CA condition. Two samples from markedly anaemic patients had low blood haemoglobin and RBC count before and after reflex. The remaining 13 samples were obtained from 12 patients, most of whom were on antiretroviral therapy or suffered severe electrolyte disorders, known conditions associated to increased MCHC. The implementation of the proposed automatic reflex by reticulocyte channel on the Sysmex XN-9000 platform in a TLA setting may solve the problem of spuriously high MCHC due to RBC agglutination for CA in a few minutes instead of waiting hours for sample preheating.

Searching for a role of procalcitonin determination in COVID-19: a study on a selected cohort of hospitalized patients.

Objectives: Procalcitonin (PCT) has been proposed for differentiating viral vs. bacterial infections. In COVID-19, some preliminary results have shown that PCT testing could act as a predictor of bacterial co-infection and be a useful marker for assessment of disease severity. Methods: We studied 83 COVID-19 hospitalized patients in whom PCT was specifically ordered by attending physicians. PCT results were evaluated according to the ability to accurately predict bacterial co-infections and death in comparison with other known biomarkers of infection and with major laboratory predictors of COVID-19 severity. Results: Thirty-three (39.8%) patients suffered an in-hospital bacterial co-infection and 44 (53.0%) patients died. In predicting bacterial co-infection, PCT showed a relatively low accuracy (area under receiver-operating characteristic [ROC] curve [AUC]: 0.757; 95% confidence interval [CI]: 0.651-0.845), with a strength for detecting the outcome not significantly different from that of white blood cell count and C-reactive protein (CRP). In predicting patient death, PCT showed an AUC of 0.815 (CI: 0.714-0.892), not better than those of other more common laboratory tests, such as blood lymphocyte percentage (AUC: 0.874, p=0.19), serum lactate dehydrogenase (AUC: 0.860, p=0.47), blood neutrophil count (AUC: 0.845, p=0.59), and serum albumin (AUC: 0.839, p=0.73). Conclusions: Procalcitonin (PCT) testing, even when appropriately ordered, did not provide a significant added value in COVID-19 patients when compared with more consolidated biomarkers of infection and poor clinical outcome. The major application of PCT in COVID-19 is its ability, associated with a negative predictive value >90%, to exclude a bacterial co-infection when a rule-out cut-off (<0.25 µg/L) is applied.

Is a pharmacogenomic panel useful to estimate the risk of oxaliplatin-related neurotoxicity in colorectal cancer patients?

Oxaliplatin-induced peripheral neurotoxicity (OXPN) is a dose-limiting toxicity in colorectal cancer (CRC) patients. Single nucleotide polymorphisms (SNPs) in genes involved in drug transport may lead to higher intracellular oxaliplatin accumulation in the dorsal root ganglia and thus increased risk of OXPN. In this study, a panel of 5 SNPs, namely ABCC2 (-24C > T/rs717620 and c.4544 G > A/rs8187710), ABCG2 (c.421 C > A/rs2231142), ABCB1 (c.3435 C > T/rs1045642) and SLC31A1 (c.-36 + 2451 T > G/rs10981694), was evaluated to assess their association with grade 2-3 OXPN in metastatic CRC patients. SNPs were considered according to a dominant model (heterozygous + homozygous). Germline DNA was available from 120 patients who received oxaliplatin between 2010 and 2016. An external cohort of 80 patients was used to validate our results. At the univariable logistic analyses, there were no significant associations between SNPs and incidence of OXPN. Taking into account the strength of observed association between OXPN and the SNPs, a clinical risk score was developed as linear predictor from a multivariable logistic model including all the SNPs together. This score was significantly associated with grade 2-3 OXPN (p = 0.036), but the external calibration was not satisfactory due to relevant discrepancies between the two series. Our data suggest that the concomitant evaluation of multiple SNPs in oxaliplatin transporters is an exploratory strategy that may deserve further investigation for treatment customization in CRC patients.

Clinical and genetic factors associated with increased risk of severe liver toxicity in a monocentric cohort of HIV positive patients receiving nevirapine-based antiretroviral therapy.

BACKGROUND: Nevirapine has been used as antiretroviral agent since early '90. Although nevirapine is not currently recommended in initial anti-HIV regimens, its use remains consistent in a certain number of HIV-1-positive subjects. Thus, our aim was to determine clinical and genetic factors involved in the development of severe nevirapine induced liver toxicity. METHODS: We retrospectively analyzed all HIV positive patients who were followed at the Infectious Diseases Unit, DIBIC Luigi Sacco, University of Milan from May 2011 to December 2015. All patients treated with nevirapine who underwent a genotyping for the functional variants mapping into ABCB1, CYP2B6, CYP3A4 and CYP3A5 genes were included in the analysis. Severe hepatotoxicity was defined as ACTG grade 3-4 AST/ALT increase during the first three months of nevirapine treatment. The causality assessment between NVP exposure and drug-induced liver injury was performed by using the updated Roussel Uclaf Causality Assessment Methods. Hardy Weinberg equilibrium was tested by χ2 test. A multivariable logistic regression model was constructed using a backward elimination method. RESULTS: Three hundred and sixty-two patients were included in the analysis, of which 8 (2.2%) experienced a severe liver toxicity. We observed no differences between patients with and without liver toxicity as regards gender, ethnicity, age and immune-virological status. A higher prevalence of HCV coinfection (75.0% vs 30.2%; p = .0013) and higher baseline AST (58 IU/L vs 26 IU/L; p = 0.041) and ALT (82 IU/L vs 27 IU/L; p = 0.047) median levels were observed in patients with liver toxicity vs those without toxicity. The genotypes CT/TT at ABCB1 rs1045642 single nucleotide polymorphism (SNP), showed a protective effect for liver toxicity when compared with genotype CC (OR = 0.18, 95%CI 0.04-0.76; p = 0.020) in univariate analysis. In the multivariate model, HCV coinfection was independently associated with higher risk of developing liver toxicity (aOR = 8.00, 95%CI 1.27-50.29; p = 0.027), whereas ABCB1 rs1045642 CT/TT genotypes (aOR = 0.10, 95%CI 0.02-0.47; p = 0.004) was associated with a lower risk. CONCLUSIONS: According to our findings HCV coinfection and ABCB1 rs1045642 SNP represent independent determinants of severe liver toxicity related to nevirapine. This genetic evaluation could be included as toxicity assessment in HIV-1-positive subjects treated with nevirapine.

Renal function in HIV/HBV co-infected and HBV mono-infected patients on a long-term treatment with tenofovir in real life setting.

The human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection is likely to be associated with an increased risk of kidney disease, due to the additional factors that may affect renal function in the HIV population. We aimed to evaluate renal toxicity in HIV/HBV and HBV mono-infected patients on long-term therapy with tenofovir (TDF) and to explore the association of polymorphisms in ATP-binding cassette (ABCC)2, ABCC4, ABCC10 with the development of renal dysfunction. From September 2006 to November 2014, 44 HIV/HBV co-infected and 34 HBV mono-infected patients were commenced on TDF. Data of renal safety were retrospectively collected and analyzed. ABCC2, ABCC4 and ABCC10 genotypes were identified by real-time PCR. Over 60 months of observation, there was a significant increase in mean creatinine levels from baseline (P<.01) that was not significantly different between the two study groups. Moreover, a significant decline in estimated glomerular filtration rate (eGFR) was observed from baseline (P<.01), and it was significantly greater in HBV mono-infected than co-infected patients (P=.03). The distribution of ABCC2, ABCC4 and ABCC10 genotypes among a subgroup of 34 patients did not show significant association with eGFR decline <90 mL/min per 1.73 m2 . Although our findings showed a statistically significant decrease in eGFR with long-term use of TDF, its clinical impact seems to be modest. The role of genetic factors to identify patients at greater risk for developing tenofovir-induced renal toxicity needs to be further investigated.

Fluoropyrimidine-Associated Toxicity in Two Gastrointestinal Cancer Patients: Potential Role of Common DPYD Polymorphisms.

While the majority of patients can be treated safely with fluoropyrimidine, some experience severe fluoropyrimidine-associated toxicity. The frequency and severity of these adverse events vary from patient to patient and are partially explained by genetic polymorphism into the dihydropyrimidine dehydrogenase (DPYD) gene. Carriers of the rare allelic variants DPYD*2A, DPYD*13, and DPYD D949V are more likely to experience severe adverse reactions during fluoropyrimidine-based therapy. However, these 3 genetic variants explain only a small percentage of the overall drug toxicity, and more frequent ones such as homozygous or compound heterozygous DPYD V732I can play a key role.

SLC29A1 polymorphism and prediction of anaemia severity in patients with chronic hepatitis C receiving triple therapy with telaprevir.

OBJECTIVES: The equilibrative nucleoside transporter 1 (ENT1) is the main protein involved in ribavirin cellular uptake. Polymorphisms at the SLC29A1 gene, encoding ENT1, may influence ribavirin-associated anaemia, which is observed at a higher incidence with telaprevir in combination with pegylated-IFNα and ribavirin than with pegylated-IFNα and ribavirin alone. In this study, we investigated the role of the rs760370 SLC29A1 variant in ribavirin-induced anaemia in chronic hepatitis C patients treated with telaprevir-based triple therapy. METHODS: Forty patients infected with hepatitis C virus (HCV) genotype 1 and starting anti-HCV therapy with telaprevir in combination with pegylated-IFN/ribavirin were prospectively evaluated for SNPs at the SLC29A1 gene and inosine triphosphatase (ITPA) genes using a real-time PCR system. RESULTS: 40% of patients developed severe anaemia with a haemoglobin (Hb) decline ≥ 5 g/dL from the pretreatment value. The SLC29A1 rs760370 GG genotype was associated with the severity of Hb decrease as expressed by the median (IQR) Hb nadir change from baseline [-5.4 (-5.6; -5.0) g/dL in GG versus -4.2 (-5.1; -3.4) in AA/AG genotype; P=0.05] and by the Hb decrease ≥ 5 g/dL by week 12 (77.8% of GG carriers versus 24% of AA/AG; P<0.01). In multivariate analysis, older age (P=0.03), lower baseline Hb concentration (P=0.02) and SLC29A1 rs760370 GG (P=0.02) were associated with the development of severe anaemia during treatment, whereas no association was found with ITPA SNPs in our population receiving telaprevir-based therapy. CONCLUSIONS: In patients with chronic hepatitis C receiving telaprevir-based therapy, SNP rs760370A>G at the SLC29A1 gene influences the severity of ribavirin-induced anaemia, possibly mirroring the erythrocyte uptake of ribavirin.

DPD and UGT1A1 deficiency in colorectal cancer patients receiving triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan.

AIMS: Triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan is a standard therapy for metastatic colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) in DPYD and UGT1A1 influence fluoropyrimdines and irinotecan adverse events (AEs). Low frequency DPYD variants (c.1905 + 1G > A, c.1679 T > G, c.2846A > T) are validated but more frequent ones (c.496A > G, c.1129-5923C > G and c.1896 T > C) are not. rs895819 T > C polymorphism in hsa-mir-27a is associated with reduced DPD activity. In this study, we evaluated the clinical usefulness of a pharmacogenetic panel for patients receiving triplet combinations. METHODS: Germline DNA was available from 64 CRC patients enrolled between 2008 and 2013 in two phase II trials of capecitabine, oxaliplatin and irinotecan plus bevacizumab or cetuximab. SNPs were determined by Real-Time PCR. We evaluated the functional variants in DPYD (rare: c.1905 + 1G > A, c.1679 T > G, c.2846A > T; most common: c.496A > G, c.1129-5923C > G, c.1896 T > C), hsa-mir-27a (rs895819) and UGT1A1 (*28) genes to assess their association with grade 3-4 AEs. RESULTS: None of the patients carried rare DPYD variants. We found DPYD c.496A > G, c.1129-5923C > G, c.1896 T > C in heterozygosity in 19%, 5% and 8%, respectively, homozygous rs895819 in hsa-mir-27a in 9% and homozygous UGT1A1*28 in 8%. Grade 3-4 AEs were observed in 36% patients and were associated with DPYD c.496A > G (odds ratio (OR) 4.93, 95% CI 1.29, 18.87; P = 0.021) and homozygous rs895819 in hsa-mir-27a (OR 11.11, 95% CI 1.21, 102.09; P = 0.020). Carriers of DPYD c.1896 T > C and homozygous UGT1A1*28 showed an OR of 8.42 (95% CI 0.88, 80.56; P = 0.052). Multivariate analysis confirmed an independent value for DPYD c.496A > G and c.1896 T > C. CONCLUSIONS: Concomitant assessment of DPYD variants and the UGT1A1*28 allele is a promising strategy needing further validation for dose personalization.

ITPA and SLC29A1 Genotyping for the Prediction of Ribavirin Dose Reduction in Anti-HCV Triple Therapy with Protease Inhibitors.

Chronic hepatitis C is one of the most important causes of liver disease, leading to cirrhosis, hepatic decompensation and hepatocellular carcinoma. Recently some important advances in therapy have been achieved with the introduction of first wave, first generation direct acting antiviral agents (DAAs) such as boceprevir (BOC), in combination with pegylated interferon (Peg-IFN) and ribavirin (RBV). The superior rate of sustained virological response with this treatment is accompanied by an elevated frequency of anaemia. Several studies have evidenced the importance of single nucleotide polymorphisms (SNPs) in inosine triphosphatase (ITPA) and solute carrier family 29, member 1 (SLC29A1) genes in the development of this adverse event. Here, we investigated haemoglobin levels and the best-known functional SNPs in ITPA and SLC29A1 genes in 22 patients treated with triple therapy with BOC/Peg-IFN/RBV. The identification of ITPA protective and SLC29A1 risk genotypes still appears to be a current methodology in RBV dosing during hepatitis C virus therapy with DAAs.

Undetected toxicity risk in pharmacogenetic testing for dihydropyrimidine dehydrogenase.

Fluoropyrimidines, the mainstay agents for the treatment of colorectal cancer, alone or as a part of combination therapies, cause severe adverse reactions in about 10%-30% of patients. Dihydropyrimidine dehydrogenase (DPD), a key enzyme in the catabolism of 5-fluorouracil, has been intensively investigated in relation to fluoropyrimidine toxicity, and several DPD gene (DPYD) polymorphisms are associated with decreased enzyme activity and increased risk of fluoropyrimidine-related toxicity. In patients carrying non-functional DPYD variants (c.1905+1G>A, c.1679T>G, c.2846A>T), fluoropyrimidines should be avoided or reduced according to the patients' homozygous or heterozygous status, respectively. For other common DPYD variants (c.496A>G, c.1129-5923C>G, c.1896T>C), conflicting data are reported and their use in clinical practice still needs to be validated. The high frequency of DPYD polymorphism and the lack of large prospective trials may explain differences in studies' results. The epigenetic regulation of DPD expression has been recently investigated to explain the variable activity of the enzyme. DPYD promoter methylation and its regulation by microRNAs may affect the toxicity risk of fluoropyrimidines. The studies we reviewed indicate that pharmacogenetic testing is promising to direct personalised dosing of fluoropyrimidines, although further investigations are needed to establish the role of DPD in severe toxicity in patients treated for colorectal cancer.

Seizures in patients with chronic hepatitis C treated with NS3/4A protease inhibitors: does pharmacological interaction play a role?

The addition of NS3/4A protease inhibitors boceprevir and telaprevir to pegylated interferon (Peg-IFN)-α and ribavirin for the treatment of hepatitis C virus (HCV) genotype 1-infected patients has led to higher rates of virological response and adverse events. Among the several side effects of interferon, neuropsychiatric symptoms have been described, particularly depression and anxiety, occurring in about 25% of patients. Although seizures have been reported in interferon-treated patients with multiple sclerosis and in a variety of malignancies, the epileptogenic potential of interferon-α in the treatment of HCV infection is considered minimal. In this report we present a new onset of seizures occurring in 2 patients during anti-HCV therapy in association with Peg-IFN, ribavirin and HCV protease inhibitors.

Corrigendum: Definition of the immune parameters related to COVID-19 severity.

[This corrects the article DOI: 10.3389/fimmu.2022.850846.].

Definition of the Immune Parameters Related to COVID-19 Severity.

A relevant portion of patients with disease caused by the severe acute respiratory syndrome coronavirus 2 (COVID-19) experience negative outcome, and several laboratory tests have been proposed to predict disease severity. Among others, dramatic changes in peripheral blood cells have been described. We developed and validated a laboratory score solely based on blood cell parameters to predict survival in hospitalized COVID-19 patients. We retrospectively analyzed 1,619 blood cell count from 226 consecutively hospitalized COVID-19 patients to select parameters for inclusion in a laboratory score predicting severity of disease and survival. The score was derived from lymphocyte- and granulocyte-associated parameters and validated on a separate cohort of 140 consecutive COVID-19 patients. Using ROC curve analysis, a best cutoff for score of 30.6 was derived, which was associated to an overall 82.0% sensitivity (95% CI: 78-84) and 82.5% specificity (95% CI: 80-84) for detecting outcome. The scoring trend effectively separated survivor and non-survivor groups, starting 2 weeks before the end of the hospitalization period. Patients' score time points were also classified into mild, moderate, severe, and critical according to the symptomatic oxygen therapy administered. Fluctuations of the score should be recorded to highlight a favorable or unfortunate trend of the disease. The predictive score was found to reflect and anticipate the disease gravity, defined by the type of the oxygen support used, giving a proof of its clinical relevance. It offers a fast and reliable tool for supporting clinical decisions and, most important, triage in terms of not only prioritization but also allocation of limited medical resources, especially in the period when therapies are still symptomatic and many are under development. In fact, a prolonged and progressive increase of the score can suggest impaired chances of survival and/or an urgent need for intensive care unit admission.

ABCB1 c.3435C>T polymorphism is associated with platinum toxicity: a preliminary study.

BACKGROUND: Platinum-based doublets are the standard chemotherapy for lung cancer. The identification of markers associated with drug toxicity may improve the success of the treatment. Single nucleotide polymorphisms (SNPs) mapping into the genes involved in platinum transport or detoxification may explain the occurrence of toxicities. In this study, we evaluated the role of three SNPs in predicting the onset of adverse events for lung cancer patients receiving cisplatin or carboplatin in adjuvant, neo-adjuvant and metastatic settings. METHODS: Eighty-two patients affected by non-small-cell and small-cell lung cancer treated with cisplatin- or carboplatin-based chemotherapy (stage II-IV) were enrolled. Before genetic analysis, patients signed a written informed consent. DNA was extracted from peripheral blood samples and genotypes were determined by real-time PCR. We selected and analyzed three SNPs: ABCB1 c.3435C>T/rs1045642, ABCC2 -24C>T/rs717620 and GSTP1 c.313A>G/rs1695. Patient characteristics and genotypes were correlated with hematological, gastrointestinal and renal toxicity as recorded by Common Terminology Criteria for Adverse Event (CTCAE) v4.03. No neurological toxicity was observed in our patients. RESULTS: Variant alleles were present in 53% of patients for ABCB1 c.3435C >T, 18.3% for ABCC2 -24C> T, and 34.8% for GSTP1 c.313A>G. Heterozygous CT at ABCB1 c.3435 was associated to a lower risk of hematological toxicity compared to homozygous CC (OR = 0.20; 95% CI 0.05, 0.69; p = 0.01). Similar results were observed by genetic dominant model (CT + TT vs CC) and hematological toxicity (OR = 0.26; 95% CI 0.09, 0.79; p = 0.02). No other significant associations were found between toxicity and SNPs. Multivariate analysis confirmed an independent value for the ABCB1 c.3435 C >T polymorphism. CONCLUSIONS: The present study reveals that ABCB1 c.3435C>T polymorphism influences platinum toxicity. The T allele seems to exert a protective effect on the development of toxicities. Further studies, such as epigenetic regulation ones, are needed to validate and shed more light on this association.