A metagenomics workflow for SARS-CoV-2 identification, co-pathogen detection, and overall diversity.

An unbiased metagenomics approach to virus identification can be essential in the initial phase of a pandemic. Better molecular surveillance strategies are needed for the detection of SARS-CoV-2 variants of concern and potential co-pathogens triggering respiratory symptoms. Here, a metagenomics workflow was developed to identify the metagenome diversity by SARS-CoV-2 diagnosis (npositive = 65; nnegative = 60), symptomatology status (nsymptomatic = 71; nasymptomatic = 54) and anatomical swabbing site (nnasopharyngeal = 96; nthroat = 29) in 125 individuals. Furthermore, the workflow was able to identify putative respiratory co-pathogens, and the SARS-CoV-2 lineage across 29 samples. The diversity analysis showed a significant shift in the DNA-metagenome by symptomatology status and anatomical swabbing site. Additionally, metagenomic diversity differed between SARS-CoV-2 infected and uninfected asymptomatic individuals. While 31 co-pathogens were identified in SARS-CoV-2 infected patients, no significant increase in pathogen or associated reads were noted when compared to SARS-CoV-2 negative patients. The Alpha SARS-CoV-2 VOC and 2 variants of interest (Zeta) were successfully identified for the first time using a clinical metagenomics approach. The metagenomics pipeline showed a sensitivity of 86% and a specificity of 72% for the detection of SARS-CoV-2. Clinical metagenomics can be employed to identify SARS-CoV-2 variants and respiratory co-pathogens potentially contributing to COVID-19 symptoms. The overall diversity analysis suggests a complex set of microorganisms with different genomic abundance profiles in SARS-CoV-2 infected patients compared to healthy controls. More studies are needed to correlate severity of COVID-19 disease in relation to potential disbyosis in the upper respiratory tract. A metagenomics approach is particularly useful when novel pandemic pathogens emerge.

Donor-Recipient Matching for KIR Genotypes Reduces Chronic GVHD and Missing Inhibitory KIR Ligands Protect against Relapse after Myeloablative, HLA Matched Hematopoietic Cell Transplantation.

BACKGROUND: Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT. METHODS AND FINDINGS: The study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into 'discovery' (135 pairs) and 'validation' (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50-5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33-5.11). High incidence of cGVHD associated with KIR genotype mismatching was applicable to both sibling and unrelated donors and was specific to recipients who had one or two C1 bearing HLA-C epitopes (HLA-C1/x, p = 0.001; SHR = 2.40; 95%CI: 1.42-4.06). When compared with KIR genotype mismatched transplants, HLA-C1/x patients receiving grafts from KIR genotype matched donors had a significantly improved cGRFS (p = 0.013; HR = 1.62; 95%CI: 1.11-2.39). Although there was no effect of KIR genotype matching on survival outcomes, a significantly reduced incidence of relapse (p = 0.001; SHR = 0.22; 95%CI: 0.10-0.54) and improved relapse-free survival (p = 0.038; HR = 0.40; 95%CI: 0.17-0.95) was observed with one or more missing ligands for donor inhibitory KIR among the recipients of unrelated donor transplants. CONCLUSIONS: The present study for the first time presents the beneficial effects of KIR genotype matching in reducing cGVHD in myeloablative transplant setting using HLA matched (sibling and unrelated) donors. The findings offer a clinically applicable donor selection strategy that can help control cGVHD without affecting the risk of relapse and/or identify patients at a high risk of developing cGVHD as potential candidates for preemptive therapy. The findings also affirm the beneficial effect of one or more missing inhibitory KIR ligands in the recipient in reducing relapse and improving a relapse free survival in unrelated donor transplants.

Killer immunoglobulin like receptor gene content diversity among Northern Indian population.

BACKGROUND: Genes encoding KIR receptors are clustered in one of the most variable regions of the human genome. KIR gene frequencies vary in worldwide populations and reveal high probability of individuals differing in their gene content. AIM: This study aimed to investigate KIR diversity among the northern Indian population who share features with either Western Eurasian or East Asian populations. It sought to decipher how northern Indians are associated phylogenetically with global populations whilst also focusing on differentiation of populations. SUBJECTS AND METHODS: This paper studied 867 northern Indians using PCR-SSP. Gene and genotypic frequencies were calculated, using statistical analyses. Findings were compared against 76 global populations of differing ethnicities. RESULTS: This northern Indian population shared characteristics with Western Eurasian or Asian Indian populations, as is evident from genetic distance, clustered heatmap, phylogenetic assessment and principal component analysis. The findings are consistent with the demographic history of northern India, including specific features, such as presence of comparatively high KIR B-haplotype as compared to A-haplotype. CONCLUSION: KIR frequencies and profiles of northern Indians were more similar to Western Eurasians, Africans and Asian Indians. This may suggest that KIR genes are under constant evolutionary pressures and selection, which may be linked to different invading pathogens.

Genetic association of adipokine and UCP2 polymorphism with recurrent miscarriage among non-obese women.

The adipokines produced from adipose tissues influence energy homeostasis, resulting in alterations of the adipokine concentrations. This process may be associated with fertility impairment, resulting in recurrent miscarriage. The present study investigated whether there was any association between the UCP2 45-bp indel polymorphism and the adipokine gene polymorphisms, namely leptin 2549 (C/A), adeponectin 276 (G/T) and 45 (T/G) and resistin 420 (C/G) in 200 non-obese recurrent miscarriage patients and 300 ethnically matched negative controls. These markers were studied using gene-specific PCR single specific primer and restriction fragment length polymorphism. For leptin 2549 and adeponectin 276, the A allele and G allele showed 3.42-fold (P=0.0001) and 1.36-fold (P=0.036) increased risk of recurrent miscarriage, respectively. Combined analysis of UCP2 45-bp indel and leptin 2549 showed U0-L0 and U1-L0 variants to be at 2- and 3-fold increased associative risk, respectively. Combined analysis of leptin 2549 and adeponectin 276 showed L0-D0 and L0-D1 variants to be at 2- and 4-fold increased associative risk, respectively. The combination U1-L0-D1-A1-R1 was 4.39-fold higher (P=0.0007) among recurrent miscarriage patients. In conclusion, the results highlight the role of the studied adipokine and UCP2 polymorphisms in recurrent miscarriage among the North Indian non-obese population. Pregnancy invokes a large shift in maternal metabolism. The normal concentrations of adipokines, which maintain the integrity of the hypothalamus-pituitary-gonadal axis, regular ovulatory processes and successful embryo implantation, are altered because of the influence of energy homeostasis, which in turn leads to fertility impairment and recurrent miscarriage of unknown aetiology. Recurrent miscarriage is reported in higher frequency among obese women. The UCP2 45-bp indel polymorphism and the adipokine gene polymorphisms namely leptin 2549 (C/A), adeponectin 276 (G/T), adeponectin 45 (T/G) and resistin 420 (C/G) have been shown to be associated with obesity. Most of the adipokine-related studies done previously have taken into consideration the metabolic function and obesity. However, there exist very few studies to evaluate the role of adipokines in non-obese recurrent miscarriage with no cause of repeated pregnancy losses. The present study focused at evaluating the independent effect of these single-nucleotide polymorphisms in non-obese women undergoing recurrent miscarriage.

Upregulation of HLA-G in JEG-3 cells by dexamethasone and hydrocortisone.

BACKGROUND: Various reports suggest that HLA-G molecule plays an important role in feto-maternal interface, protecting the allogenic fetus from maternal immune attack. It is shown that steroid hormones may upregulate the HLA-G gene expression. In the present study, we have made an attempt to upregulate the HLA-G gene expression in a HLA-G(+ve) cell line (JEG-3) by using two glucocorticoids drugs, i.e., dexamethasone and hydrocortisone. METHODS: Choriocarcinoma JEG-3 (HLA-G(+ve)), JAR (HLA-G(-ve)) and erythroleukemia K-562 (HLA-G(-ve)) cell lines were obtained from American Type Culture Collection. These cell lines were treated with glucocorticoids (dexamethasone and hydrocortisone). HLA-G gene transcription was determined by standard and real-time RT-PCR analysis, and protein expression was evaluated by both flow cytometry and Western blotting. RESULTS: Dose-dependent increase in HLA-G mRNA and protein expression was observed in HLA-G(+ve) JEG-3 cells, while no expression was recorded in JAR and K-562 (HLA-G(-ve)) cell lines. CONCLUSION: We were able to upregulate HLA-G expression only in HLA-G(+ve) cell line. On the basis of our results, we hypothesize that the HLA-G gene expression can be upregulated only when the cell lines/cells have the basal expression and not in the cells that totally lack its expression. We have further hypothesized that these drugs may be used only in those women with recurrent miscarriages who show minimum basal expression level of HLA-G.

Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.

The association of four common polymorphisms of vascular endothelial growth factors (VEGF) with recurrent miscarriages(RM) was evaluated in North Indian women for 200 patients with RM and 200 controls. The subjects were genotyped for the polymorphisms 2578C/A, 2549 18-bp I/D, 1154G/A and +936C/T. Association of VEGF genotypes, alleles and haplotypes with recurrent miscarriage were evaluated by Fisher's exact test. 1154G/A and +936C/T modified the risk of RM. The 1154A allel and +936T allel significantly increased the risk of RM (OR = 1.485, P = 0.0210, 95% CI 1.072­2.057 and OR = 1.869, P = 0.0054, 95% CI 1.214­2.876 respectively). Risk was further increased when ­1154A/A genotype and +936C/T genotype were considered (OR = 2.0, P = 0.0310,95% CI 1.068­3.747 and OR = 1.716, P = 0.0293, 95% CI 1.058­2.784 respectively). However, no association was found between 2578C/A or 2549 18-bp I/D and RM. Four haplotypes, AIAC, ADAC, CIAT and ADGT, were found to predispose to RM while the haplotypes CIAC, CDGT and ADGC were found to show protective effect. In conclusion, two common polymorphisms of the VEGF gene,1154G/A and +936C/T, increase the risk of RM in North Indian women. RM is also predisposed in the presence of haplotypes AIAC,ADAC, CIAT and ADGT.

Recurrent pregnancy loss and apolipoprotein E gene polymorphisms: a case­control study from north India.

PROBLEM: the role of apolipoprotein E gene polymorphisms in the etiology of recurrent pregnancy loss (RPL) is not clearly understood. We evaluated this polymorphism in unexplained pregnancy losses among North Indian women. METHOD OF STUDY: in a retrospective case­control study, 200 well-characterized RPL cases were examined for their APO-E genotypes based on restriction fragment length polymorphism analysis of PCR-amplified fragments including amino acid positions 112 and 158. The observed genotypes were compared with those obtained from an equal number of ethnically matched negative controls. RESULTS: we found similar APO-E genotypes and E2, E3, and E4 allele frequency distribution among RPL patients and controls. The allele frequencies obtained in patients and controls respectively were as follows:E2 = 7.5% and 9.0% (P = 0.52; OR = 0.81; 95%CI = 0.49­1.35),E3 = 89.7% and 90% (P = 1.00; OR = 0.97; 95%CI = 0.61­1.54), and E4 = 2.8% and 1% (P = 0.12; OR = 2.79; 95%CI = 0.88­8.86). CONCLUSIONS: our data did not support the association of APO-E gene polymorphisms with recurrent pregnancy loss as reported by some of the previous studies.We endorse adequate characterization of RPL cases, inclusion of appropriate negative controls, and adequate sample size prior to addressing such studies.