PCR In Peripheral Blood Research Articles

The Prevalence and Molecular Characterization of Bovine Babesia Species and the First Report of B. bovis from Kashmir Himalayas.

Bovine babesiosis, a global disease, has not been studied so far in Kashmir valley, which is having temperate type of climate as compared to rest of India having tropical to sub-tropical climate, so we felt the need to investigate it. To diagnose the babesiosis in clinically suspected cattle (n=450), peripheral blood film examination and PCR tests using generic and species-specific primers targeting Babesia/Theleria genera and B. bigemina, B. bovis as well as B. divergens, respectively were conducted. Four PCR products were sequenced and subjected to BLASTn analysis. Ticks were collected from the clinically suspected animals and identified as per the standard morphological keys. The prevalence of babesiosis among suspected cattle in central Kashmir by peripheral blood film examination and PCR technique was 11.11% and 33.62%, respectively. The 18S rRNA gene of Isolate B1 of Babesia spp. showed 99.0 to 100% nucleotide sequence homology with 18S rRNA gene of different isolates of B. bigemina registered in the GenBank, while as 18S rRNA gene of Isolate Z showed 98.5 to 99.2% and 93.1 to 93.9% nucleotide sequence homology with 18S rRNA gene of different isolates of Babesia spp. and B. bigemina, respectively, registered in the GenBank. Rhipicephalus spp. and Haemaphysalis spp. were the two major tick genera identified in the present study. Bovine Babesiosis in Kashmir is attributed to B. bovis, B. bigemina and some other Babesia spp. or strains which needs further investigation. To our knowledge, this is the first report of Babesia bovis from northern India in cattle.

A NUDT15 Genetic Polymorphism Study in Adult Acute Leukemia Patients Integrating Pharmacogenomics and Clinical Practice: A Single Centre Experience from South India

Background: Severe myelosuppression secondary to thiopurines such as 6-Mercaptopurine (6-MP) in patients with acute leukemia undergoing maintenance therapy is frequently attributed to inherited genetic polymorphisms of Thiopurine Methyltransferase (TPMT); however, this is rare in the Asian population. Nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) is also responsible for encoding critical enzymes involved in 6-MP metabolism. We aimed to determine the frequency of genetic polymorphisms of NUDT15 in the South Indian population and the impact of the omission of 6-MP from the treatment protocol of these patients. Methods: We retrospectively reviewed 17 patients diagnosed with acute leukemia between May 2018 and May 2020 and analyzed the prevalence of NUDT15 genetic polymorphisms in these patients. The NUDT15 mutation study (NUDT15 415C>T) used allele-specific Real-Time PCR in peripheral blood. 6-Mercaptopurine (6-MP) was excluded from the treatment regimen of patients with NUDT15 genetic polymorphisms and the impact of its exclusion was also studied. Results: Among the 17 patients for whom we cared, 10 were diagnosed with B-cell Acute Lymphoblastic Leukemia (B-ALL), three with T-cell Acute Lymphoblastic Leukemia (T-ALL), one with Mixed Phenotype Acute Leukemia (MPAL), and one with Acute Promyelocytic Leukemia (APML). Treatment records could not be retrieved for 2 patients. NUDT15 mutations were present in six patients (36%) (see fig1), of whom two had homozygous (33%) polymorphisms and four had heterozygous (67%) polymorphisms. In the heterozygous group, 2 were Ph. (Philadelphia chromosome) negative B-ALL; one was treated with BFM and the other with hyper-CVAD, one Ph. positive B-ALL was treated with BFM + Ponatinib, and one High-risk APML was treated with the APML 4 protocol. In the homozygous group, treatment details could not be retrieved for one case, and the other patient had T-ALL, which was treated with hyper-CVAD. 6-MP was omitted from the treatment protocol for all NUDT15 mutation-positive patients, both homozygous and heterozygous. Of these patients, two developed grade 1 myelosuppression, one developed grade 2 myelosuppression, one developed dyselectrolytemia, and one patient did not develop any toxicity. The incidence of severe or grade 3 and 4 myelosuppression in NUDT15 polymorphisms receiving 6MP can be as high as 50% (Khaeso K et.al Front Pharmacol. 2021). In comparison, none of our patients developed grade 3 or 4 myelosuppression. Overall, treatment-related adverse events included grade 1-2 myelosuppression (n=6), grade 1-2 hepatotoxicity (n=1), deep vein thrombosis (n=2), and sepsis (n=2). Five of the 6 patients with NUDT15 mutations achieved complete remission (CR). Conclusion: The prevalence of NUDT15 polymorphisms was high in our study population. Based on a meta-analysis, the prevalence of NUDT15 in the South Indian population was 16.5%, and in patients with adverse effects, the pooled prevalence of NUDT15 polymorphisms was 49.51% (Anuraag Jena et.al. Expert Review of Clinical Pharmacology 2021). Based on our results, although limited by the small number of cases, we suggest that pre-emptive testing to detect these polymorphisms should be undertaken in all adult acute leukemia patients of South Indian origin, given their high prevalence in this population. The omission of 6-MP from treatment protocols can help prevent severe myelosuppression and treatment interruption or discontinuation, which in turn can improve treatment outcomes.

Correlation of Peripheral Chimeric Antigen Receptor T-cell (CAR-T Cell) mRNA Expression Levels with Toxicities and Outcomes in Patients with Diffuse Large B-cell Lymphoma

Cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are significant complications in patients with relapsed/refractory diffuse large B-cell lymphoma undergoing chimeric antigen receptor T-cell (CAR-T cell) therapy. However, it remains unclear whether CAR-T cell expression itself is clinically relevant. We assessed CAR-T cell mRNA expression and DNA concentration by digital droplet PCR in peripheral blood from 14 sequential CAR-T cell recipients. Patients were grouped according to CAR-T cell peak expression. Patients with high CAR-T cell peak expression (8 patients; 57%) had higher rates of ICANS (p=0.0308) and intensive care unit admission (p=0.0404), longer durations of hospitalization (p=0.0077), and, although not statistically significant, a higher rate of CRS (p=0.0778). There was a correlation of CAR-T cell mRNA expression with DNA concentration, but CAR-T cell expression levels failed to correlate to response or survival. Our data suggest that higher CAR-T cell peak mRNA expression is associated with increased risk for ICANS and possibly CRS, requiring further investigation in larger studies.

Hsa_circ_0001162 Inhibition Alleviates High Glucose-Induced Human Podocytes Injury by the miR-149-5p/MMP9 Signaling Pathway.

Emerging evidences suggested that circular RNAs (circRNAs) are involved in diabetic nephropathy (DN). Accumulating evidence had suggested that the degree of podocyte is a major prognostic determinant of DN progression. However, the function and in-depth mechanisms of hsa_circ_0001162 in podocyte injury of DN remain unclear. Hsa_circ_0001162 expression was detected by real-time quantitative PCR (RT-qPCR) in peripheral blood of DN patients and high glucose-induced podocytes injury model. The cell counting kit 8, 5-ethynyl-2'-deoxyuridine, flow cytometry with Annexin V-FITC/PI staining, caspase-3 activity assay Kit, enzyme linked immunosorbent assay (ELISA), RT-qPCR and western blotting were used to evaluate the effect of hsa_circ_0001162 / miR-149-5p / MMP9 axis on high glucose-induced podocyte injury. Mechanistically,dual luciferase reporter was used to confirm the relationship of miR-149-5p and hsa_circ_0001162 or MMP9. Furthermore, RNA-pull down and immunoprecipitation assay were implemented to verify the potential regulatory effects of EIF4A3 on biogenesis of hsa_circ_0001162. Our results showed that hsa_circ_0001162 was highly expressed in peripheral blood of DN patients and high glucose-induced podocytes injury model, and the knockdown of hsa_circ_0001162 increased the proliferation, inhibited the apoptosis, and suppressed inflammatory response in high glucose-induced podocytes injury. Mechanism studies demonstrated that EIF4A3 bound with flanking sequences of hsa_circ_0001162 to promote hsa_circ_0001162 expression, upregulated hsa_circ_0001162 increased the MMP9 expression via sponging miR-149-5p, thus aggravating the high glucose-induced podocytes injury. Overall, our data demonstrated that knockdown of hsa_circ_0001162 inhibited high glucose-induced podocytes injury by regulating miR-149-5p/MMP9 axis, and intervention of hsa_circ_0001162/miR-149-5p/MMP9 axis may be a potentially promising therapeutic strategy for podocyte injury in DN patients.

DNA methylation profiling reveals novel pathway implicated in cardiovascular diseases of diabetes.

Epigenetics was reported to mediate the effects of environmental risk factors on disease pathogenesis. We intend to unleash the role of DNA methylation modification in the pathological process of cardiovascular diseases in diabetes. We screened differentially methylated genes by methylated DNA immunoprecipitation chip (MeDIP-chip) among the enrolled participants. In addition, methylation-specific PCR (MSP) and gene expression validation in peripheral blood of participants were utilized to validate the DNA microarray findings. Several aberrantly methylated genes have been explored, including phospholipase C beta 1 (PLCB1), cam kinase I delta (CAMK1D), and dopamine receptor D5 (DRD5), which participated in the calcium signaling pathway. Meanwhile, vascular endothelial growth factor B (VEGFB), placental growth factor (PLGF), fatty acid transport protein 3 (FATP3), coagulation factor II, thrombin receptor (F2R), and fatty acid transport protein 4 (FATP4) which participated in vascular endothelial growth factor receptor (VEGFR) signaling pathway were also found. After MSP and gene expression validation in peripheral blood of participants, PLCB1, PLGF, FATP4, and VEGFB were corroborated. This study revealed that the hypomethylation of VEGFB, PLGF, PLCB1, and FATP4 might be the potential biomarkers. Besides, VEGFR signaling pathway regulated by DNA methylation might play a role in the cardiovascular diseases' pathogenesis of diabetes.

Assessment of acute kidney injury in dogs suffering from babesiosis and leptospirosis

Acute kidney injury (AKI) is a serious complication of infectious diseases like babesiosis and leptospirosis in dogs. Both the diseases show similar clinical manifestations, due to which timely differential diagnosis often gets delayed. The present study focused on diagnosis of major infectious agents causing AKI and its grading in dogs. Peripheral blood smear examination and PCR were used for the diagnosis of babesiosis. Leptospirosis was screened using PCR and MAT and was later confirmed by MAT on paired sera sample. Diagnosis of AKI was made based on the increase in serum creatinine levels ≥0.3 mg/dL within 48 h of presentation. Staging of animals was carried out as per International Renal Interest Society (IRIS) guidelines. All the dogs with confirmed leptospirosis and 15 per cent of dogs with babesiosis were diagnosed with AKI. The timely diagnosis of AKI in dogs during their initial stages helps to regain normal renal function within short-term treatment itself.

Lncrna Famlf-2 Inhibits Acute Lymphoblastic Leukemia Apoptosis through the p38 MAPK Signaling Pathway

Background:Acute lymphocytic leukemia (ALL) is a hematologic malignancy caused by the clonal proliferation of immature lymphocytes. Long noncoding RNAs (lncRNAs) are involved in the pathogenesis and progression of ALL. Our previous study identified Familial acute myelogenous leukemia related factor (FAMLF) from a AML pedigree. There are at least three splice variants from FAMLF, referred as FAMLF-1, FAMLF-2 and FAMLF-3. We have shown higher expression of FAMLF-1 was related to a higher complete remission (CR) rate, and shorter relapse free survival (RFS) in AML. Another FAMLF variants, FAMLF-2 is highly expressed in ALL, and the potential mechanism of FAMLF-2 in regulating ALL progression is unclear. This study aims to investigate the functional and mechanistic roles of FAMLF-2 in ALL. Methods:The expression of FAMLF-2 was detected by real-time PCR (Q-PCR) in peripheral blood of ALL patients (N=85) and healthy controls (N=60). The full length of FAMLF-2 was obtained by 5' and 3' rapid amplification of the cDNA ends (RACE). FAMLF-2 subcellular location in Jurkat cells was detected by lncRNA FISH, nuclear/cytoplasmic RNA fractionation, and qPCR. The proliferation and apoptosis of Jurkat cell were determined by CCK-8 and flow cytometry experiments in vitro. RNA-seq was performed to study the molecular mechanism of FAMLF-2 in regulating ALL cell apoptosis. Results:The FAMLF-2 mRNA expression and in ALL patients was significantly higher than those in the normal individuals (P=0.000). The expression of FAMLF-2 was positively correlated with percentage of peripheral blood blasts (P=0.032). The ALL patients with higher FAMLF-2 expression had significantly poor overall survival (P=0.036). We defined a 2393-bp full-length transcript of FAMLF-2, and examined the subcellular localization of FAMLF-2, which showed that FAMLF-2 predominately resides in the nucleus of Jurkat cells. The cell proliferation was inhibited, and cell apoptosis was induced when FAMLF-2 were down-regulated in Jurkat and Nalm6 cells, whereas the overexpression of FAMLF-2 showed the opposite results. We analyzed the RNA-seq data of the knockdown and overexpression of FAMLF-2 in Jurkat cells, and the GSEA analysis suggested that FAMLF-2 knockdown was associated with "MYC TARGETS V1", "MYC TARGETS V2" pathways. The knockdown of FAMLF-2 in Jurkat cells upregulated the expression of p-P38 and downregulated the expression of c-Myc, bcl-2. Conclusion: Taken together, our study indicate that FAMLF-2 inhibits the apoptosis of ALL through the p38 MAPK signaling pathways, which might act as a prospective prognostic biological marker for ALL. Keywords: Acute lymphocytic leukemia, Long noncoding RNAs, FAMLF-2, apoptosis, p38 MAPK Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nasopharyngeal microbiota profiling of pregnant women with SARS-CoV-2 infection

We aimed to analyze the nasopharyngeal microbiota profiles in pregnant women with and without SARS-CoV-2 infection, considered a vulnerable population during COVID-19 pandemic. Pregnant women were enrolled from a multicenter prospective population-based cohort during the first SARS-CoV-2 wave in Spain (March-June 2020 in Barcelona, Spain) in which the status of SARS-CoV-2 infection was determined by nasopharyngeal RT–PCR and antibodies in peripheral blood. Women were randomly selected for this cross-sectional study on microbiota. DNA was extracted from nasopharyngeal swab samples, and the V3-V4 region of the 16S rRNA of bacteria was amplified using region-specific primers. The differential abundance of taxa was tested, and alpha/beta diversity was evaluated. Among 76 women, 38 were classified as positive and 38 as negative for SARS-CoV-2 infection. All positive women were diagnosed by SARS-CoV-2 IgG and IgM/IgA antibodies, and 14 (37%) also had a positive RT–PCR. The overall composition of the nasopharyngeal microbiota differ in pregnant women with SARS-CoV-2 infection (positive SARS-CoV-2 antibodies), compared to those without the infection (negative SARS-CoV-2 antibodies) (p = 0.001), with a higher relative abundance of the Tenericutes and Bacteroidetes phyla and a higher abundance of the Prevotellaceae family. Infected women presented a different pattern of microbiota profiling due to beta diversity and higher richness (observed ASV < 0.001) and evenness (Shannon index < 0.001) at alpha diversity. These changes were also present in women after acute infection, as revealed by negative RT–PCR but positive SARS-CoV-2 antibodies, suggesting a potential association between SARS-CoV-2 infection and long-lasting shift in the nasopharyngeal microbiota. No significant differences were reported in mild vs. severe cases. This is the first study on nasopharyngeal microbiota during pregnancy. Pregnant women with SARS-CoV-2 infection had a different nasopharyngeal microbiota profile compared to negative cases.

Discrete Typing Units of Trypanosoma cruzi Identified by Real-Time PCR in Peripheral Blood and Dejections of Triatoma infestans Used in Xenodiagnosis Descriptive Study.

Chagas disease (ChD) is a vector zoonosis native to the American continent caused by the protozoan parasite Trypanosoma cruzi; the biological vectors are multiple species of hematophagous insects of the family Triatominae. A relevant aspect in the host–parasite relationship is the identification of the various genotypes of T. cruzi called discrete typing units (DTU) that circulate in mammals and vectors. In Chile, it has been described that the DTUs TcI, TcII, TcV, and TcVI circulate in infected humans, vectors, and wild animals. Identifying DTUs has acquired clinical importance, since it has been suggested that different genotypes could cause distinct pathologies, circulate in different geographical areas, and present different sensitivities to trypanocidal drugs. In this study, circulating T. cruzi DTUs in peripheral blood and Triatoma infestans dejections used in xenodiagnosis (XD) were amplified by qPCR in 14 Chilean patients with chronic ChD from highly endemic areas. More positive samples were detected by XD compared to peripheral blood samples, and 64.28% of the cases were simple infections and 35.72% mixed, with a statistically significant difference in the frequency of TcV DTU. This study would suggest that T. infestans from Chile is more competent to amplify one DTU over others, probably due to a process of co-evolution.

SUCCESSFUL CLEARANCE OF CYTOMEGALOVIRUS COLITIS IN ACUTE SEVERE ULCERATIVE COLITIS DURING INDUCTION OF OZANIMOD

Abstract INTRODUCTION Cytomegalovirus (CMV) colitis, commonly observed in patients with acute severe ulcerative colitis (UC), increases the risk for colectomy and treatment resistance. Unfortunately, corticosteroids and immunomodulators increase CMV reactivation and infection. The use of certain biologics such as anti-TNF mAb increase the risk for CMV colitis, while experience with newer biologics is limited leaving clinicians with few validated options. Ozanimod, an oral sphingosine-1-phosphate receptor modulator, was recently approved for induction and maintenance therapy for UC. Experience with ozanimod in the setting of UC complicated by CMV infection has not been reported to this date. We report here on a severe UC patient with tissue proven CMV ulceration that cleared the viral infection during ozanimod induction. CASE REPORT A 21-year-old male with UC treated with tofacitinib presented with five days of diffuse abdominal pain and 10-12 episodes of bloody diarrhea per day with CRP 341mg/L (&amp;lt;8.0mg/L) and calprotectin &amp;gt;3000μg/g (&amp;lt;49μg/g). Computed topography of the abdomen and pelvis showed diffuse wall thickening and mucosal enhancement throughout the entire colon and rectum. He was started on methylprednisolone 20mg TID for acute severe UC. He previously failed infliximab, adalimumab, ustekinumab, and vedolizumab. Colonoscopy showed pancolitis (Mayo score 3) with deep ulceration in the cecum. Random colon biopsies were consistent with active colitis with an area of CMV infection in an area of cecal ulceration (Figure 1). Serum CMV Quantitative PCR was 410IU/ml (normal &amp;lt;50IU/ml). The patient refused colectomy and was started on IV ganciclovir 5mg/kg q12h while transitioning to prednisone 40mg/day. Three weeks after initial presentation and eleven days after starting ganciclovir, the patient began ozanimod. He remained on ganciclovir for three weeks then transitioned to oral valganciclovir 900mg BID. The patient’s symptoms improved, CRP normalized, and calprotectin levels dropped (1200μg/g). Repeat colonoscopy performed eighteen days after ozanimod initiation revealed moderately-improved pancolitis (Mayo 2) with multiple healing cecal ulcers. Analysis of cecal ulceration IHC and peripheral blood PCR indicated clearing of the infection from the colon (Figure 2). DISCUSSION Cytomegalovirus colitis remains a common complication in severe UC, while avoidance of colectomy with viral clearance remains a challenge. Ozanimod and ganciclovir were chosen by this patient after refusing surgical resection. The improvement in clinical biomarkers of colitis and viral clearance suggest that ozanimod given with ganciclovir may be an exciting rescue therapy in these patients. Future studies will be needed to ascertain the rates of ozanimod response and CMV clearance in a larger patient cohort.

SUCCESSFUL CLEARANCE OF CYTOMEGALOVIRUS COLITIS IN ACUTE SEVERE ULCERATIVE COLITIS DURING INDUCTION OF OZANIMOD

Cytomegalovirus (CMV) colitis, commonly observed in patients with acute severe ulcerative colitis (UC), increases the risk for colectomy and treatment resistance. Unfortunately, corticosteroids and immunomodulators increase CMV reactivation and infection. The use of certain biologics such as anti-TNF mAb increase the risk for CMV colitis, while experience with newer biologics is limited leaving clinicians with few validated options. Ozanimod, an oral sphingosine-1-phosphate receptor modulator, was recently approved for induction and maintenance therapy for UC. Experience with ozanimod in the setting of UC complicated by CMV infection has not been reported to this date. We report here on a severe UC patient with tissue proven CMV ulceration that cleared the viral infection during ozanimod induction. A 21-year-old male with UC treated with tofacitinib presented with five days of diffuse abdominal pain and 10-12 episodes of bloody diarrhea per day with CRP 341mg/L (<8.0mg/L) and calprotectin >3000μg/g (<49μg/g). Computed topography of the abdomen and pelvis showed diffuse wall thickening and mucosal enhancement throughout the entire colon and rectum. He was started on methylprednisolone 20mg TID for acute severe UC. He previously failed infliximab, adalimumab, ustekinumab, and vedolizumab. Colonoscopy showed pancolitis (Mayo score 3) with deep ulceration in the cecum. Random colon biopsies were consistent with active colitis with an area of CMV infection in an area of cecal ulceration (Figure 1). Serum CMV Quantitative PCR was 410IU/ml (normal <50IU/ml). The patient refused colectomy and was started on IV ganciclovir 5mg/kg q12h while transitioning to prednisone 40mg/day. Three weeks after initial presentation and eleven days after starting ganciclovir, the patient began ozanimod. He remained on ganciclovir for three weeks then transitioned to oral valganciclovir 900mg BID. The patient’s symptoms improved, CRP normalized, and calprotectin levels dropped (1200μg/g). Repeat colonoscopy performed eighteen days after ozanimod initiation revealed moderately-improved pancolitis (Mayo 2) with multiple healing cecal ulcers. Analysis of cecal ulceration IHC and peripheral blood PCR indicated clearing of the infection from the colon (Figure 2). Cytomegalovirus colitis remains a common complication in severe UC, while avoidance of colectomy with viral clearance remains a challenge. Ozanimod and ganciclovir were chosen by this patient after refusing surgical resection. The improvement in clinical biomarkers of colitis and viral clearance suggest that ozanimod given with ganciclovir may be an exciting rescue therapy in these patients. Future studies will be needed to ascertain the rates of ozanimod response and CMV clearance in a larger patient cohort.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Updated Results of a Phase II Study of Ponatinib and Blinatumomab for Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Updated Results of a Phase II Study of Ponatinib and Blinatumomab for Patients with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

P05 Blast transformation of essential thrombocythemia presenting as hip monoarthritis

P05 Blast transformation of essential thrombocythemia presenting as hip monoarthritis

T-cell clonality in peripheral blood as a predictor of progression to systemic treatment in patients with stage IB mycosis fungoides (MF).

e19538 Background: A major unmet clinical need in the care of early-stage MF patients is the identification of those with a high risk of failing skin directed therapy or progressing to advanced disease. Herein, we inquired if the identification of a clonal T-cell receptor (TCR) gene rearrangement by PCR in peripheral blood could predict the clinical outcome, particularly the need for systemic treatment, in patients with stage IB MF. Methods: This is a retrospective cohort study of patients with stage IB MF who underwent peripheral blood TCR clonality analysis by PCR. The primary outcome of the study was time from diagnosis to initiation of systemic treatment. Secondary outcomes were: (1) time to progression to advanced-stage disease (stages IIB-IV) and (2) overall survival. Patients were censored at time of last clinical follow up. Log rank test was used to compare the survival distributions of the two groups; p value &lt; 0.05 was considered significant. Results: From May 2014 to October 2019, 56 consecutive stage IB pts with &gt; 6 months follow up were included in this analysis. Peripheral blood TCR clonality status was available in 42 patients: 18 pts had a positive TCR clone and 24 did not. Median follow up time was 36 months (range 8.5 – 198 months). At 3 years, 39% of patients with peripheral clone had progressed to systemic treatment versus 8% of those without a peripheral clone (log rank test, p-value = 0.003). For the secondary outcomes, at 3 years 17% of patients with peripheral clone had progressed to advanced stage versus 4% of those without (log rank test, p-value = 0.10); 5% of patients with peripheral clone had died versus 0% of those without (log rank test, p-value = 0.03). Conclusions: Detection of a predominant TCR clone by PCR in the peripheral blood is an important prognostic marker in the initial workup of MF, as its presence is highly correlated with subsequent progression to systemic treatment and death. If this finding is validated, it can be used to risk stratify and individualize therapy for MF patients.[Table: see text]

CYP3A5 and PPARA genetic variants are associated with low trough concentration to dose ratio of tacrolimus in kidney transplant recipients.

Genetic polymorphisms have been associated with variation in the metabolism of tacrolimus (TAC) in kidney transplant patients. This study is aimed at assessing the impact of allelic variants of CYP3A5 and PPARA genes on the pharmacokinetics (PK) of TAC in Brazilian kidney transplant recipients in the first-year post-transplant. A total of 127 patients were included for genetic evaluation. Genomic DNA was isolated from peripheral blood and real-time PCR was used to analyze the main polymorphisms described for the genes CYP3A5 (rs776746; C> G) and PPARA (rs4823613; A> G and rs4253728; G> A). CYP3A5 expressors showed a lower Co/dose ratio than non-expressors, with the median values of this parameter <1.01ng/mL/mg in the first group at all evaluated times. Additionally, PPARA variant homozygotes had a lower Co/D ratio than wild allele carriers in the 12-month post-transplant period, with a median value of 0.65ng/mL/mg. In the CYP3A5 expressers, the presence of the variant homozygous genotype PPARA was associated with a lower value of Co/D compared with the other genotypic groups at month 12. In the population under study, polymorphisms on CYP3A5 and PPARA were identified as determining and independent factors associated with the reduction of Co/D of TAC. Thus, the genotyping of these genetic variants may be a useful tool for the individualized prescription of TAC in kidney transplant patients.

Incidence, Risk Factors, and Outcomes of Viral Reactivations in Alternative Donor Haematopoietic Stem Cell Transplant

Introduction : There has been an increase in number of alternative donor [Unrelated donor (UD) or Haploidentical (HIT)] transplants in recent years due to better graft vs. host disease (GVHD) prophylaxis and comparable outcomes with matched sibling transplants. However, viral reactivations frequently complicate these transplants and can lead to organ dysfunction, poor graft function and death. We aimed to analyze incidence, risk factors, and outcomes of viral reactivations in alternative donor transplants at our centre. Methods: All UD or HIT transplants performed between January 1st 2009 and February 29th 2020 were included in this retrospective analysis. Conditioning regimen used were either myeloablative (TBI based) or reduced intensity (fludarabine based). All patients received T cell replete grafts. Calcineurin inhibitors (with methotrexate or mycophenolate mofetil) were used for GVHD prophylaxis. Rabbit anti-thymocyte globulin (ATG) was used in UD transplants and post transplant cyclophosphamide was used in HIT. Cytomegalovirus (CMV) was monitored twice weekly from start of conditioning. Adenovirus (ADV), BK, Epstein Barr virus (EBV) were monitored weekly post engraftment. Monitoring was done by real time quantitative PCR in peripheral blood for CMV, EBV and in blood as well as urine for ADV and BK virus. Parvovirus and Human Herpes virus-6 (HHV-6) were monitored if clinically indicated. Viral reactivation was defined as 2 consecutive readings of &amp;gt;150 copies/mL. Monitoring was stopped after withdrawal of immunosuppressants and restarted with onset of GVHD or use of systemic steroids. Ganciclovir (GCV) was usually preferred for pre-emptive treatment of CMV. Cidofovir (CDV), foscarnet (FOS), artesunate (ART) or leflunomide (LEF) were used when GCV failed or was myelotoxic. Cidofovir was used for ADV disease. Intravenous immunoglobulin (IVIg) prophylaxis was used in all patients. Primary objective was to estimate the incidence of viral reactivation. Secondary objectives were to explore various risk factors associated with viral reactivations and determine their strength of association using odds ratio (OR). Results: Of 334 allogeneic transplants performed during this period, 87 were alternative donor transplants (26 UD and 61 HIT). Incidence of CMV reactivation was 80.5 % (92 episodes of CMV reactivations in 70 patients). Two in UD transplant and 4 in HIT developed CMV colitis. Acute GVHD was a significant risk factor for developing CMV reactivation (OR=4.32, 95% CI 1.3-13.6; p=0.013). Pre-emptive 1st line treatment with GCV was used in 59 episodes, ART in 10, LEF in 19, CDV and FOS was used in 1 and 3 episodes each. No deaths were attributed to CMV disease. Incidence of ADV reactivation was 26% (21 episodes in 80 patients tested). Four patients developed ADV disease and were treated with CDV (ADV colitis in 2, ADV cystitis in 1, and 1 had colitis with CNS involvement) of which 2 died. Use of ATG (OR 3.5, 95% CI 1.2 - 10; p=0.03) and presence of acute GVHD (OR 7.9, 95% CI 2.5- 25; p=0.0004) were significant risk factors of ADV reactivation. EBV reactivation was seen in 25 of 68 patients tested (incidence 36.7%).Two patients developed post transplant lymphoproliferative disorder requiring therapy with rituximab, of which 1 died. One out of 15 patients tested by qualitative PCR had HHV 6 reactivation and primary graft failure. BK viral reactivation was seen in 59 of 71 tested (incidence 83.1%). Grade I and II hemorrhagic cystitis (Droller's grading system) was seen in 12 and 11 patients respectively. Foley's catheterization with bladder irrigation was used in 3 patients and intravesical GCSF was given to 1 patient. Parvovirus infection was seen in 2 of 12 patients tested, of which 1 was given IVIg. Cumulative incidence of viral reactivations is shown in Figure 1. Viral reactivation characteristics and risk factors are shown in tables 1 and 2 respectively. Median OS for the cohort of 87 patients was 52 months. There was no statistically significant impact of viral reactivations on OS. Conclusion: Incidence of CMV reactivation is high (80%) in alternative donor HSCT and acute GVHD is a significant risk factor for CMV, ADV and EBV reactivation. Use of ATG is a significant risk factor for ADV reactivation. However, rates of ADV, BK virus, and EBV reactivation requiring intervention were low. Better prophylactic strategies are necessary for prevention of CMV reactivations in alternative donor transplants. Disclosures No relevant conflicts of interest to declare.

Ockham\u2019s razor defeated: about two atypical cases of hemolytic uremic syndrome

BackgroundMedical investigation is a favorite application of Ockham’s razor, in virtue of which when presented with competing hypotheses, the solution with the fewest assumptions should be privileged. Hemolytic uremic syndrome (HUS) encompasses diseases with distinct pathological mechanisms, such as HUS due to shiga-like toxin-producing bacteria (STEC-HUS) and atypical HUS, linked to defects in the alternate complement pathway. Other etiologies such as Parvovirus B19 infection are exceptional. All these causes are rare to such extent that we usually consider them mutually exclusive. We report here two cases of HUS that could be traced to multiple causes.Cases presentationCase 1 presented as vomiting and diarrhea. All biological characteristics of HUS were present. STEC was found in stool (by PCR and culture). After initial remission, a recurrence occurred and patient was started on Eculizumab. Genetic analysis revealed the heterozygous presence of a CFHR1/CFH hybrid gene. The issue was favorable under treatment.In case 2, HUS presented as fever, vomiting and purpura of the lower limbs. Skin lesions and erythroblastopenia led to suspect Parvovirus B19 primo-infection, which was confirmed by peripheral blood and medullar PCR. Concurrently, stool culture and PCR revealed the presence of STEC. Evolution showed spontaneous recovery.ConclusionsBoth cases defy Ockham’s razor in the sense that multiple causes could be traced to a single outcome; furthermore, they invite us to reflect on the physiopathology of HUS as they question the classical distinction between STEC-HUS and atypical HUS. We propose a two-hit mechanism model leading to HUS. Indeed, in case 1, HUS unfolded as a result of the synergistic interaction between an infectious trigger and a genetic predisposition. In case 2 however, it is the simultaneous occurrence of two infectious triggers that led to HUS. In dissent from Ockham’s razor, an exceptional disease such as HUS may stem from the sequential occurrence or co-occurrence of several rare conditions.

“Miro” Study, a FIL multicenter phase II trial combining local radiotherapy and MRD-driven immunotherapy in early-stage follicular lymphoma.

e20073 Background: Early stage follicular lymphoma (FL) is usually managed with involved field radiotherapy (IFRT), allowing eradication of the disease in 40-50% of patients (pts). The aim of this multicenter phase II prospective study was to evaluate the role of minimal residual disease (MRD) in identifying pts unlikely to be cured by IFRT, for whom an immunotherapy-based consolidation could improve outcome. Methods: 110 pts with stage I/II FL were enrolled and treated with 24 Gy IFRT. Peripheral blood (PB) and bone marrow (BM) samples were centralized to the FIL (Federazione Italiani Linfomi) MRD Network of EuroMRD-certified laboratories. In BCL2/IGH+ pts at baseline, MRD was analyzed by nested PCR (NEST) and RQ-PCR (RQ) in BM and/or PB after IFRT and every 6 months over a 3-year period. Pts with positive MRD by both NEST and RQ in the BM and/or PB after IFRT or who became positive during the follow-up (FU) were treated with 8 weekly doses of the anti-CD20 MoAb ofatumumab (OFA). Primary objective: efficacy of immunotherapy in obtaining a negative MRD. Results: 49 of 106 pts were males. Median age was 55 y (29-83). 69% of pts had inguinal involvement. At baseline, 30% of pts had a BCL2/IGH rearrangement (30 MBR, 1 MBR and mcr, 1 mcr) in BM and/or PB; the concordance between compartments was 90%. All pts obtained a clinical response after IFRT, with persistence of BCL2/IGH+ cells in PB and/or BM in 60% of cases. MRD-positive pts, either after IFRT (n = 18) or in case of conversion to a positive signal during the FU (n = 7), received OFA, obtaining a conversion to MRD negativity, in 20/22 cases (91% - CI 65.1-97.1), significantly superior to the expected 50%. After a median FU of 25 months, all but 2 pts who achieved a negative MRD with OFA are still negative. A clinical relapse or progression was observed in 17 pts: 13 (18%) among the 73 “no marker” pts; 2 relapses (16%) were observed among the 12 MRD-negative pts after IFRT and 2 among the 25 treated with OFA (8%), 1 having achieved a MRD negativity and 1 not. No significant differences in PFS have so far been observed between the three groups. Conclusions: In early stage FL RT alone is often insufficient to eradicate the disease, inducing a negative MRD only in 40% of pts, long-lasting only in half of them. The primary objective of this study - MRD negativity after immunotherapy - was achieved. The strategy of an immunotherapy consolidation after IFRT in MRD-positive pts allowed to increase molecular responses. A longer FU and further studies will allow to define the impact of this MRD-driven strategy also on clinical outcome. Clinical trial information: EudraCT number: 2012-001676-11 .

Association of CYP11B2 gene polymorphism with preeclampsia in north east of Iran (Khorasan province)

PurposeIdentification the genetic factors in preeclampsia (PE) are useful to increase the current knowledge of the pathophysiology of the disorder. The genetic factors implicated for all cases of PE remain to be determined. This study was aimed to investigate association between ADD1 1378G > T, AGTR2 1675G > A, AGTR1 1166A > C, NOS3 894 G > T and CYP11B2 −344C > T gene polymorphisms in Iranian women with PE. Material and Methods117 pregnant women with PE and 103 healthy women without affected previous pregnancy by PE were selected. Genomic DNA was extracted from peripheral blood and real-time PCR was performed to investigate the polymorphisms using a commercial kit. ResultsThere was a significant difference in CYP11B2 −344C > T gene polymorphism between case and control groups (P = 0.025). The odds ratio was 0.71 (CI 95% = 0.28–1.79). There were no statistical significant differences between other genetic polymorphisms. ConclusionOur results showed a significant association between CYP11B2 −344C > T gene polymorphism with PE. This finding suggests that mentioned polymorphism may be associated with susceptibility to PE at least in IRAN.

Monoclonal Gammopathy with Secondary Atypical Hemolytic-Uremic Syndrome

Monoclonal gammopathy is a clonal plasma cell disorder associated with an increased risk of progression to lymphoproliferative malignancy. In children, monoclonal and oligoclonal gammopathies are rare, and their clinical relevance unrecognized. Atypical hemolytic uremic syndrome (aHUS) is a rare endotheliopathy associated with dysregulated activation of complement, microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. We present a case of aHUS that we hypothesize was triggered by monoclonal gammopathy in a child several years after cardiac transplantation. An 11 year old African-American girl with sickle trait was transferred to our cardiac center with a two week history of epigastic pain, emesis, microcytic anemia, hypergammaglobulinemia, and development of renal failure requiring dialysis. She had undergone cardiac transplantation at 7 months of age for dilated cardiomyopathy of unknown etiology and had recovered from acute cardiac rejection requiring ECMO at age 5 years. She had previously experienced at least two episodes of acute kidney injury requiring dialysis with full recovery of function reported. Renal biopsy revealed a severe collapsing glomerulopathy and a thrombotic microangiopathy. Upon transfer to our institution, her labs were notable for microcytic anemia, normal platelet count, haptoglobin, and LDH. A week later, she developed aHUS diagnosed by hemoglobin 9.5 gm/dL, haptoglobin &lt; 30 mg/dL, LDH 314 U/L, platelets 100,000, and a predominance of schistocytes on peripheral blood smear in the setting of acute renal failure. Serum protein electrophoresis (SPEP) revealed a monoclonal spike (1g/dL), identified as IgG kappa by immunoabsorption. A second, smaller monoclonal spike was identified as IgA lambda. aHUS evaluation revealed low C3, normal C4, and 50% CD46 expression by flow cytometry. Bone marrow biopsy with flow cytometry excluded lymphoproliferative disorders. EBV did not amplify with quantitative PCR of peripheral blood, and computed tomography imaging of the body did not detect abnormal mass or lymph nodes. A trial of plasmapheresis did not improve biochemical and hematologic features of aHUS. A trial of eculizumab resulted led to normalization of platelet count and C3, increased haptoglobin, reduced need for red cell transfusion, and less frequent dialysis. Monoclonal gammopathy of unknown significance (MGUS) is rare in children, but may be more common in transplant recipients, especially after cardiac transplants in infants when thymectomy is performed. MGUS associated with secondary aHUS has been rarely reported in adults. The mechanism for aHUS was demonstrated in one case to result from affinity of the paraprotein to a binding site on complement factor H, with subsequent loss of factor H cofactor activity to inhibit alternative complement activation. We hypothesize that immune dysregulation from fluctuating levels of paraproteins during inflammation in our patient with monoclonal gammopathy resulted in recurrent, intermittent episodes of aHUS. We speculate that this was responsible for the patient's collapsing glomerulopathy and dialysis-dependent chronic renal failure. This case highlights the importance of considering monoclonal gammopathy in pediatric patients who develop recurrent kidney failure or atypical hemolytic uremic syndrome in the setting of immune dysregulation. The merit of treating the monoclonal gammopathy with Bortezumib and Rituximab is currently under consideration. Disclosures No relevant conflicts of interest to declare.