Detection of clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex and Klebsiella pneumoniae isolated from farmed freshwater fish.

AIMS: The present study was aimed to detect clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex (CR-ECC), Klebsiella pneumoniae (CR-KP), and Serratia plymuthica (CR-SP) isolated from farmed freshwater fish. METHODS AND RESULTS: Out of 243 spatially diverse freshwater fish samples analysed, 5.3% were contaminated with CR-ECC, 1.6% with CR-KP, and 0.4% with CR-SP. The CR-ECC was further identified as E. asburiae (38.5%), E. mori (23.1%), E. cloacae (15.4%), E. hormaechei (15.4%), and E. kobei (7.7%) by 16S rRNA gene sequencing. The CR-ECC were resistant to carbapenems and cefoxitin, whereas CR-KP and CR-SP were multi-drug resistant (MDR). The CR-ECC harboured the carbapenemase gene blaIMI alone or in combination with blaTEM, blaEBC, blaCIT, blaACC, and tet(E). Whereas, CR-KP harboured carbapenemase gene, blaNDM-5 along with blaOXA-48, blaSHV, blaOXA-1, blaCTX-M-15, tet(A), sul1, and qnrB. No carbapenemase-encoding genes were detected in CR-SP. The MLST analysis showed that CR-KP belonged to ST231 and ST1561 lineages, while CR-ECC did not show exact match with any reported STs. The plasmid replicons predominantly detected were IncF and IncI1. Broth mating assays of CR-KP and CR-ECC with recipient Escherichia coli J53 indicated that blaNDM-5 was transferable but not blaIMI. CONCLUSION: This study highlights the low-level contamination of carbapenem-resistant Enterobacterales (CRE) harbouring clinically relevant carbapenemase-encoding genes in farmed freshwater fish from India. The CR-ECC of fish origin did not show the potential to spread carbapenem resistance.

Safety and efficacy of Sofosbuvir and Velpatasvir in children with active hepatitis C virus infection undergoing haploidentical transplantation.

Direct Acting Antivirals (DAA) have changed the landscape of hepatitis C virus (HCV) infection with high cure rates across genotypes. However, the use of these agents in the setting of allogeneic hematopoietic cell transplantation (HCT) has been limited. In this context, we report the outcome of five children (5-12 years) with relapsed and refractory leukemia and active HCV infection (genotype 1b), who underwent urgent haploidentical HCT and were treated with Sofosbuvir and Velpatasvir (Sof-Vel) from initiation of treatment to 24 weeks post-HCT. All achieved complete virologic response (VR) at a median of 2 weeks, with normalization of liver enzymes. There were no adverse events related to the use of Sof-Vel, with no major fluctuations in cyclosporine levels. Two of the patients developed chronic GVHD and one relapsed. Sof-Vel was continued in one of them along with sirolimus without affecting drug levels. With a median follow-up of 15 months, four patients are disease free with sustained VR. Our study shows that combination of Sof-Vel might be effective in inducing rapid complete and sustained VR during HCT without any major untoward drug interaction.

CTLA4Ig in an Extended Schedule along with Sirolimus Improves Outcome with a Distinct Pattern of Immune Reconstitution Following Post-Transplantation Cyclophosphamide-Based Haploidentical Transplantation for Hemoglobinopathies.

The major hindrances to the success of a haploidentical hematopoietic cell transplantation for hemoglobinopathies are graft failure, early post-transplant hemophagocytic syndrome (PTHPS), and graft-versus-host disease (GVHD). Following the successful incorporation of CTLA4Ig (abatacept) in post-transplantation cyclophosphamide-based haploidentical transplantation, we piloted this approach in 10 patients (aged 3 to 19 years), with thalassemia major (TM, n=5) and sickle cell disease (n = 5). Pretransplant immunosuppressive therapy (pTIST) was administered for 10 weeks. Conditioning was myeloablative. CTLA4Ig was administered every 2 weeks during pTIST and on days -1, +5, +20, and +35 and every 4 weeks thereafter for 6 months, along with sirolimus. A short course of low-dose dexamethasone was given from day +6 for 14 days. Nine patients engrafted at a median of 15 days, with 1 patient with TM dying of sepsis on day +19. None of the patients developed acute or chronic GVHD. All 9 patients are alive and disease free at a median follow-up of 28 months. Only 4 patients had cytomegalovirus reactivation. The pattern of immune reconstitution showed a prompt and sustained recovery of T cell subsets with memory phenotype, along with early and sustained increase of Tregs and NKG2C+ natural killer (NK) cells. This novel approach, targeting CD80 and CD86 on monocytes/macrophages, promoted engraftment and limited early-onset PTHPS and graft failure. The lack of GVHD and serious infections with this approach reflects an early recovery of Tregs, memory T cells, and persistence of NKG2C+ NK cells.

Prophylactic oseltamivir during major seasonal influenza H1N1 outbreak might reduce both H1N1 and associated pulmonary aspergillosis in children undergoing haploidentical transplantation.

Following a major seasonal outbreak of H1N1 influenza in 2018 September, prophylactic oseltamivir for six months was initiated in children undergoing haploidentical HCT with regular monitoring for influenza and other respiratory virus infections. Influenza was not detected in 22 children undergoing prophylaxis, compared to 8 H1N1 infections in 21 adults without prophylaxis (P = .01). Four children on prophylaxis were detected to have other respiratory viruses, compared to 8 in those without prophylaxis. Invasive pulmonary aspergillosis (IPA) was observed only in association with H1N1 (4/8 with H1N1 vs 0/35 without H1N1, P = .001) and was thus lower in the prophylaxis group (P = .04). The overall incidence of episodes of respiratory illness and hospital stay were also lower in those on prophylaxis (P = .001). There were no untoward side effects associated with prophylactic oseltamivir. Prophylactic oseltamivir was safe and effective in prevention of H1N1 infection and subsequent IPA in children at-risk, early after haploidentical HCT.

Real world outcome of B ALL with t (1; 19) (q23; p13)/TCF3::PBX1 in adolescents and adults treated with intensive regimes.

Significant heterogeneity has been reported in outcome of Acute lymphoblastic leukemia with t(1;19)(q23;p13)/TCF3::PBX1 in adolescents and adults leading to a lack of consensus on precise risk stratification. We evaluated clinical outcome of 17 adult ALL cases (≥15 years) with this genotype treated on intensive regimes.13/17 received COG0232 and 4/17 cases received UK-ALL protocol. All achieved CR (100%) with above treatment. End of induction MRD was evaluated in 14/17 cases of which 11 (78.5%) achieved MRD negativity. Total nine patients relapsed (7 marrows, 2 CNS). Overall survival at 2 years was 53.3%. The 2 year estimated PFS was 42.9%. The 2 years CIR was 54.2%. Adults with this genotype perform poorly despite early favorable response. Incorporation of novel immunotherapies and prompt HSCT should be strongly considered with this genotype. Targeted NGS panels for additional genetic aberrations can further help in risk stratifying and guiding therapy for this genotype.

Molecular characterization of carbapenem resistant E. coli of fish origin reveals the dissemination of NDM-5 in freshwater aquaculture environment by the high risk clone ST167 and ST361.

Aquatic environment can act as reservoir and disseminator of antimicrobial resistance and resistant pathogens. Novel high-risk carbapenem resistant E. coli (CREC) are continuously emerging worldwide; however, the occurrence of CREC in freshwater aquaculture environment is largely unexplored. To fill this gap, large scale sampling of freshwater pond sites and retail fish markets was done between Oct 2020 and Oct 2021 to investigate the CREC contamination in fish. The frequency of CREC contamination in the freshwater fish was 6.99% (95% CI: 3.78-10.20%). All the isolates were MDR and harbored carbapenemase encoding gene, blaNDM-5 along with other antimicrobial resistance genes (ARGs), blaTEM (64.7%), blaCTX-M-15 (35.3%), blaOXA-1 (5.9%), tet(A) (100%), sul1 (94.1%), qnrS (82.3%), cat1 (35.3%), and cat2 (23.5%). The isolates belonged to phylogroup C and showed low virulence gene profile. ERIC-PCR grouped the isolates into five clusters (I-V). The isolates of clusters I, II, and III were identified as ST167 (76.4%) and of cluster IV as ST361 (17.6%). This is the first report documenting the contamination of NDM-5 producing E. coli ST167 and ST361 of clinical/livestock lineage in freshwater fish from India. The blaNDM-5 was significantly associated with ARGs, tet(A), and sul1; and plasmid replicons, IncF, IncI1, and IncP, signifying the presence of blaNDM-5 and associated ARGs on these transferable plasmids. These findings were validated by the successful conjugal transfer of blaNDM-5 and associated ARGs into non-CREC strain (J53). Our study highlights the ability of CREC to disseminate antimicrobial resistance which has health implications and environmental concerns.

Impact of adaptive natural killer cells, KLRC2 genotype and cytomegalovirus reactivation on late mortality in patients with severe COVID-19 lung disease.

OBJECTIVE: SARS-CoV-2 infection results in severe lung disease in up to 50% of hospitalised patients. The aetiopathogenesis in a subset of such patients, who continue to have progressive pulmonary disease following virus clearance, remains unexplored. METHODS: We investigated the role of NKG2C+/NKG2A- adaptive natural killer (ANK) cells, KLRC2 genotype and cytomegalovirus (CMV) reactivation in 22 such patients. RESULTS: The median duration of virus positivity was 23 days, and the median duration of hospitalisation was 48 days. The overall survival at 60 days in this group was 50%. Older age and comorbidities impacted survival negatively. CMV viraemia was documented in 11 patients, with a survival of 25% vs 80% in those without viraemia with viral load correlating with mortality. Both NK and T cells were markedly depressed in all patients at day 15. However, only persistently low ANK cells at 30 days along with an inversely high NKG2C-/NKG2A+ inhibitory NK cells significantly correlated with high CMV viraemia and mortality, irrespective of KLRC2 genotype. However, day 30 ANK cells were significantly lower in the KLRC2 deletion group. The release of IFN-γ and perforin was severely compromised in all patients at day +15, with significant improvement in the survivors at day +30, but not in those with adverse outcome. CONCLUSION: Patients with progressive lung disease even after negative SARS-CoV-2 status, with persistently reduced and functionally compromised ANK cells, are more likely to have CMV reactivation and an adverse outcome, independent of KLRC2 genotype.

Early and Sustained Expansion of Adaptive Natural Killer Cells Following Haploidentical Transplantation and CTLA4Ig-Primed Donor Lymphocyte Infusions Dissociate Graft-versus-Leukemia and Graft-versus-Host Effects.

BACKGROUND: Adaptive or memory natural killer (NK) cells with epigenetic imprints similar to memory T cells have been shown to develop in response to cytomegalovirus (CMV) infection with upregulation of activating receptor NKG2C. These cells have been shown to possess strong anti-tumour efficacy both in-vitro as well as in-vivo. OBJECTIVES: To determine if reconstitution of adaptive NK cells (CD56dimNKG2C+NKG2A-) in patients with advanced leukemia undergoing haploidentical HCT had any impact on disease progression (DP). STUDY DESIGN: The study cohort comprised of 60 patients with advanced acute leukemia, aged 2-65 years, receiving myeloablative PTCy based haploidentical transplantation from CMV seropositive donors, followed by CTLA4Ig-primed donor lymphocyte infusions (DLI). They were evaluated for the kinetics of reconstitution of adaptive NK cells, both phenotypic and functional, at days +30,+60, +90 and at regular intervals, to 3 years of follow-up, in relation to DP. Reconstitution of adaptive NK cells was compared with a retrospective cohort of patients in the same protocol receiving DLI without CTLA4Ig. RESULTS: Non-relapse mortality, acute and chronic GVHD were 5.1%, 10.3% and 14.5%. DP was 17.5% at a median follow-up of 28 months. Adaptive NK cells were significantly higher in patients without DP at days+30, +60 and +90 (p = 0.0001), irrespective of CMV reactivation and remained elevated until 36 months post-HCT. These cells maintained their functional competence as measured by robust interferon-gamma production with higher expressions of KIR, NKG2D and CD57, without any increase in PD1 expression. Grafts from donors with higher adaptive NK cells were associated with a lower risk of DP (p = 0.0001). In multivariate analysis, adaptive NK cell recovery at day +90 had the most favorable impact on DP (HR-0.7). Tregs reconstituted briskly along with the adaptive NK cells and were sustained as well, without compromising the GVL effect. Comparison with a retrospective cohort receiving the same protocol with DLI without CTLA4Ig, showed a superior reconstitution of adaptive NK cells in those receiving CTLA4Ig-DLI (p < 0.0001). CONCLUSION: Our study suggests that myeloablative transplantation from CMV seropositive haploidentical donors augmented with CTLA4Ig-primed DLI might favor early and sustained expansion of functionally competent adaptive NK cells irrespective of CMV reactivation, with a favorable outcome.

CTLA4Ig-primed donor lymphocyte infusions following haploidentical transplantation improve outcome with a distinct pattern of early immune reconstitution as compared to conventional donor lymphocyte infusions in advanced hematological malignancies.

CTLA4Ig has a unique property to spare or even potentiate natural killer (NK) cell-mediated cytotoxicity, whilst inhibiting T cell activation. We explored the efficacy of prophylactic DLI following CTLA4Ig (CTLA4Ig-DLI group, n = 75), compared to conventional DLI (DLI group, n = 50), in patients with advanced hematological malignancies receiving PTCy-based haploidentical transplantation. Acute and chronic GVHD in the CTLA4Ig-DLI group were 9.6% and 15.3% compared to 18.8% [p = 0.09] and 36.5% [p = 0.01] in the DLI group. Both non-relapse mortality (4% vs 14.4%) and disease progression (DP) (15.7% vs 31.1%) were lower in CTLA4Ig-DLI group (p = 0.04). GVHD and progression-free survival was significantly improved in the CTLA4Ig-DLI group (p = 0.001). The recovery of CD56dimNK cells, NKG2A-KIR + NK subsets and Tregs was significantly better in the CTLA4Ig-DLI group at all time points and memory T cells at day +90. Immune recovery in relation to DP showed distinct patterns, with T cell subsets in the DLI group and NKG2A-KIR+NK cells in CTLA4Ig-DLI group having favorable impact. CTLA4Ig-DLI was thus associated with an improved outcome, possibly on account of the distinct pattern of immune recovery shown with this novel approach.

Coexistence of giant cell fibroblastoma and encephalocele.

Giant cell fibroblastoma (GCF) is a rare soft tissue tumour that occurs almost exclusively in children younger than 10 years of age and is mostly located in the superficial soft tissues of the back and thighs. We present a rare case of GCF with encephalocele in a 1.5-year-old boy who presented with a swelling in the occipital area of the scalp since birth. CT scan suggested encephalocele without any suspicion of a mass lesion. On histopathology, an ill-defined proliferation of fibroblasts in a heavily collagenised and focally myxoid stroma was seen containing numerous multinucleated cells having a floret-like appearance along with mature glial tissue bordering a cystic space. Immunohistochemically, the stromal cells were positive for both, vimentin (diffuse) and CD34 (focal) thereby confirming the histological diagnosis of GCF. This case highlights the unusual coexistence of GCF with congenital defects and its histogenetic resemblance to dermatofibrosarcoma protuberans.

White-blue pyelocalyceal cyst with hydrotic glomerulonephritis.

A 5-month-old male infant presented with a 15 day history of distension of abdomen. On clinical examination, a soft lump was palpable in the left lumbar region. Radiological findings suggested an enlarged non-functional left kidney with ureteropelvic adhesive obstruction. The left renal mass was excised and submitted for histopathological examination. The excised renal mass was cystic with its wall partly white and partly blue. Gross and histopathological findings were diagnostic of a white-blue pyelocalyceal cyst with hydrotic glomerulonephritis. This entity needs to be differentiated from a large number of other cystic diseases of the kidney. Intrauterine screening and diagnosis may be significant for a possible early intrauterine uro-laparoscopic recanalisation of the pyeloureteral obstruction to save the affected kidney.