Ocular conjunctival microbiome profiling in dry eye disease: A case control pilot study

Purpose: Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is a multifactorial disease that results in discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. A pilot study was undertaken to determine if there were any major substantial differences in the ocular microbiome in DED patients versus healthy controls. Methods: The bacterial communities residing in the conjunctiva of patients with DED (n = 4) and healthy controls (n = 4) were assessed by 16S ribosomal RNA (rRNA) gene sequencing of the V4–V5 region. Results: The phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were most dominant and accounted for 97% and 94.5% of all bacterial sequences in patients and controls, respectively. At the genus level, 27 bacterial genera were found with more than two?fold difference between patients and controls. Four of these – Acinetobacter, Corynebacterium, Lactobacillus, and Pseudomonas spp. – dominated the ocular microbiome of all subjects, but were proportionately lower in DED (16.5%) compared to controls (37.7%). Several bacterial genera were found to be unique in DED (34) and controls (24). Conclusion: This pilot study is an attempt to profile the ocular microbiome in patients with DED that demonstrated a higher concentration of microbial DNA compared to controls, with Firmicutes phyla dominating the bacterial population in patients with DED.

Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal andoropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2aclade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were alsodetected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz.G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutationsreside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain,which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observedbetween these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations ofmiRNA binding and structure were also predicted for these mutations. More importantly, the possibleimplications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of Sprotein have also been discussed. Results report for the first time a bird’s eye view on the accumulation ofmutations in SARS-CoV-2 genome in Eastern India.

Evaluation of prolonged febrile illness in elderly

Background: Febrile illness in elderly patients in hospitals is a challenge to the physician for diagnosis and treatment due to high morbidity as well as mortality and it increases if the febrile illness is prolonged. So proper evaluation and effective management is necessary for a better outcome. Keeping in mind the scarcity of studies in elderly febrile illness in India this study was taken up.Method: A prospective study was designed in medical ICU of S.C.B Medical college and Hospital, Cuttack Odisha, India. 50 patients were included in this study from July 2007 to December 2008. Institutional Ethics Committee cleared the study.Results: In 50 elderly (Age>60 yrs) patients of prolonged febrile illness, 36 (72%) were male and 14 (28%) were female. All had fever for >21 days. Pallor was the commonest sign (62%). 30 patients had infectious etiology, 15 had malignancies. Tuberculosis was the commonest infection (28%) comprising of 46.66% of infectious etiology with Pulmonary Tuberculosis (PTB) in 20% and Extrapulmonary Tuberculosis (ETB) in 26.66%. Malignancies accounted for 30% of cases with Non-Hodgkin’s lymphoma (NHL) in 33.33% being the commonest amongst the malignancies. On follow up of 50 patients 21 (42%) got cured.Conclusion: Febrile illness in elderly needs carefully evaluation as infections account for most of the cases and Tuberculosis in our part of India as a major cause in these patients is treatable. Malignancies remain the second most common cause where timely intervention goes a long way in reducing morbidity and mortality.

Application of SNaPshot for analysis of thiopurine methyltransferase gene polymorphism.

Background & objectives: Mercaptopurine, azathioprine, and thioguanine, used as antineoplastic agents and immunosuppressants are catabolized by thiopurine methyltransferase (TPMT) enzyme, which exhibits genetic polymorphism. Genotyping patients and the population to which the patients belong, is important for effective treatment and reducing toxicity. There is a need for faster methods for genotyping. Hence the present study was planned to test the application of SNaPshot technique for analysis of the three common TPMT alleles: TPMT*2, TPMT*3A, and TPMT*3C in DNA from healthy Indian volunteers as well as to apply the method on cDNA samples obtained from children with acute lymphoblastic leukaemia (ALL). Method: A total of 120 healthy volunteers and 25 patients were analysed by multiplexed SNaPshot reaction. Genomic DNA was the template for most of the analyses, but additionally the cDNA synthesized for translocation detection was used as the template in case of patients with ALL. The results of SNaPshot reaction were confi rmed by direct sequencing. Results: The TPMT genotype could be reliably identifi ed by SNaPshot analysis in multiplex reactions both in genomic DNA samples and cDNA. The overall frequency of the three common polymorphisms was observed to be 4.9 per cent, arising from heterozygosity for TPMT*3C (4.1%) and TPMT*3A (0.8%). Interpretation & conclusion: SNaPshot method for TPMT polymorphism analysis works faster with the potential for high throughput. By simultaneously interrogating the genotype at multiple sites, the method can provide future opportunity to multiplex, though multiplexing has not been done in the present analysis. Heterozygosity for TPMT*3C (719 A>G) was detected in 4.1 per cent of the study population and no homozygosity was observed. Our results indicated that TPMT*3C was the most common polymorphism in Indian population, while TPMT3*A, associated with the absence of catalytic activity of TPMT, was very rare.