Rhinosinus Mucormycosis with Drug-induced Pancytopenia in an Immunocompromised Severe COVID-19 Patient: A Success.

Secondary infections in coronavirus disease (COVID) are becoming common. We report a case of a female known case of diabetes, sarcoidosis on steroids and methotrexate admitted with COVID pneumonia. She was treated with steroids, remdesivir, and anticoagulants and was discharged. She revisited the hospital after 2 months with complaints of severe right-sided headache, eye pain, and vomiting. Magentic resonance image of brain and paranasal sinus revealed possibility of invasive rhinosinus mucormycosis. Functional endoscopic sinus surgery (FESS) was done and culture showed growth of mucor and methicillin resistant staphylococcus aureus (MRSA) following which she was started on amphotericin B and antibiotics. She also developed methotrexate and amphotericin B-induced pancytopenia for which injection folinic acid, granulocyte-colony stimulating factor (G-CSF), and erythropoietin were given and was switched over to liposomal amphotericin B. After 5 days of ventilatory support, she was discharged in a stable condition. Extensive steroids in an immunocompromised patient might have led to this event hence physicians should always keep this possibility of secondary fungal infection in COVID patients for understanding the impact of disease. How to cite this article: Mahajan A, Tandon VS. Rhinosinus Mucormycosis with Drug-induced Pancytopenia in an Immunocompromised Severe COVID-19 Patient: A Success. Indian J Crit Care Med 2022;26(3):395-398.

C allele of rs479200 of the host EGLN1 gene - a risk factor for severe COVID-19 (pilot study)

BackgroundCoronavirus disease-2019 (COVID-19) symptoms can range from asymptomatic, moderate to severe manifestations that result in an overall global case fatality rate of 2-7 %. While each variant has had it challenges, and some variants are more severe than others, risk factors of severe COVID-19 are still under investigation. In this context, the host genetic predisposition is also a crucial factor to investigate. In the present study, we investigated host genotypes of the SNP rs479200 of the host EGLN1 gene, previously implicated in high altitude pulmonary edema (HAPE), some of whose symptoms such as hypoxia profoundly overlap with severe COVID-19. MethodsAfter informed consent, 158 RT-PCR confirmed COVID-19 patients (March 2020 to June 2021) were enrolled in the study. Based on their clinical manifestations, disease severity was categorized by the clinical team. Blood samples were drawn and DNA was extracted from the clot to infer different genotypes of the SNP rs479200 of the host EGLN1 gene. PCR-RFLP analysis of the SNP rs479200 (C > T) was performed with an amplicon size of 367 bp. Various genotypes (TT, TC and CC) were assigned based on the presence/absence of a restriction site (T/GTACA) for restriction enzyme BsrGI. Allele frequencies, Hardy-Weinberg Equilibrium (HWE) and multinomial logistic regression were performed using statistical tool SPSS version 23 (IBM). FindingsWe observed that the severe COVID-19 category was composed of comparatively younger patients with mean age (34.9{+/-}15.6), compared to asymptomatic and moderate categories whose mean age was 49.7{+/-}17.9 and 54.3{+/-}15.7, respectively. Preponderance of males and high heterozygosity (TC) was observed across the clinical categories. Notably, the frequency of C allele (0.664) was 2-fold higher than the T allele (0.336) in severe COVID-19 patients, whereas the allele frequencies were similar in asymptomatic and moderate category of COVID-19 patients. Multinomial logistic regression showed an association of genotypes with increasing clinical severity; odds ratio (adjusted OR-11.414 (2.564-50.812)) and (unadjusted OR-6.214 (1.84-20.99)) for the genotype CC in severe category of COVID-19. Interestingly, the TC genotype was also found to be positively associated with severe outcome (unadjusted OR-5.816 (1.489-22.709)), indicating association of C allele in imparting the risk of severe outcome. InterpretationThe study provides strong evidence that the presence of C allele of SNP (rs479200) of the EGLN1 gene associates with severity in COVID-19 patients. Thus, the presence of C allele may be a risk factor for COVID-19 severity. This study opens new avenues towards risk assessment that include EGLN1 (rs479200) genotype testing and identifying patients with C allele who might be prioritized for critical care.

Cellular and Antibody Immunity after COVID-19 Vaccination at >4-Month Follow Up in Immunocompetent and Immunocompromised Subjects

We evaluated post-vaccination immunity after COVID-19 vaccination with serial changes in cellular and antibody responses to the spike protein S, its S2 component which is conserved between SARS-CoV-2 and human coronaviruses, and the S1 component, which is specific to SARS-CoV-2 and also contains its receptor binding domain (RBD). In 21 healthy immunocompetent subjects all of whom demonstrated circulating IgG antibodies 4 months after mRNA1273 or BNT162b vaccination, a) the strength of S-IgG was stable while RBD-IgG declined, b) S2-reactive B-cell frequencies increased progressively (p=0.002) c) S1-reactive CD8+T-cells and CD19+B-cells were undetectable after a transient increase, and d) monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSC, PMN-MDSC) increased after the first vaccine dose. Compared with 4-month measurements from immunocompetent subjects, single samples from 20 vaccinated immunocompromised (IC) subjects revealed a) circulating S-IgG and RBD-IgG in 13 (65%) and 9 (45%) subjects, respectively, b) no differences in S2-reactive T- and B-cells, c) undetectable S1-reactive T- and B-cells, and d) fewer S-reactive CD8+T-cells and CD19+B-cells (p<0.05). Among 11 IC recipients who failed to make RBD-IgG, frequencies of PMN-MDSC were significantly higher (p<0.0004) compared with IC or immunocompetent subjects with RBD-IgG. COVID-19 vaccination induces stable antibodies to the spike protein and expands circulating B-cells reactive to the conserved spike protein sequence in immunocompetent subjects. MDSC which are known to suppress T- and B-cells, and which increase after vaccination, may limit post-vaccination responses especially among immunocompromised subjects. Antibody and cellular responses to SARS-CoV-2-specific spike antigenic sequences appear to be less durable.

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

Genetic Polymorphisms in VIR Genes among Indian Plasmodium vivax Populations

The vir genes are antigenic genes and are considered to be possible vaccine targets. Since India is highly endemic to Plasmodium vivax, we sequenced 5 different vir genes and investigated DNA sequence variations in 93 single-clonal P. vivax isolates. High variability was observed in all the 5 vir genes; the vir 1/9 gene was highly diverged across Indian populations. The patterns of genetic diversity do not follow geographical locations, as geographically distant populations were found to be genetically similar. The results in general present complex genetic diversity patterns in India, requiring further in-depth population genetic and functional studies.

Ophthalmoplegic migraine in a child, an accelerated clinical and radiologic response to steroid therapy

Ophthalmoplegic migraine is characterized by recurrent attacks of migraine-like headache with paresis of ocular cranial nerves. To date, the exact etiology of ophthalmoplegic migraine remains unknown. We report a 9-year-old girl with typical clinical features of ophthalmoplegic migraine. She presented to us shortly after onset of her fi fth episode. The initial episodes of opthalmoplegia used to last for about 2-3 months with gradual and complete recovery. Brain MRI with contrast study revealed a thickened, enhancing right oculomotor nerve in the cisternal segment during the acute phase of ophthalmoplegia. She was treated with steroid for two weeks as well as with divalproex sodium for prophylaxis of migraine. There was complete recovery of ophthalmoplegia after four weeks of treatment with complete resolution of third nerve enhancement on repeat imaging. There were no further episodes of ophthalmoplegia within a follow up period of one year. Steroid therapy may hasten the recovery of ophthalmoplegia and prophylactic treatment of migraine may reduce the episodes and severity of ophthalmoplegic migraine.

NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications.

BACKGROUND: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetics procedure for identifying point mutations in selected gene(s) amplified from a mutagenized population using high-throughput detection platforms such as slab gel electrophoresis, capillary electrophoresis or dHPLC. One essential pre-requisite for TILLING is genomic DNA isolation from a large population for PCR amplification of selected target genes. It also requires multiplexing of genomic DNA isolated from different individuals (pooling) in typically 8-fold pools, for mutation scanning, and to minimize the number of PCR amplifications, which is a strenuous and long-drawn-out work. We describe here a simplified procedure of multiplexing, NEATTILL (Nucleic acid Extraction from Arrayed Tissue for TILLING), which is rapid and equally efficient in assisting mutation detection. RESULTS: The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato. CONCLUSION: NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations.

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data.

Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides.