Molecular characterization of Ehrlichia canis from naturally infected dogs reveals a novel Asiatic-lineage and co-circulation of multiple lineages in India.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is an important rickettsial pathogen of dogs transmitted by Rhipicephalus sanguineus sensu lato ticks in India. Globally, molecular characterization of E. canis is done using different E. canis gene targets. This study aimed to characterize genetic diversity of uncultured Ehrlichia species from dogs by 16S rRNA and partial gp200 gene (termed as p43 region) sequences data. Phylogeny based on 16S rRNA gene did not reveal any region-specific lineages. The phylogeny based on 5' region of E. canis gp200 gene (termed as p43 region) revealed four major clusters (A, B, C and D) and the Indian isolates fall under clusters A and B. Cluster A is characterized by an insertion of unique 141 bp tandem repeat sequence. Similar tandem repeat sequence was found in one of the E canis isolates from east-Asia, suggesting a possible divergence within this species. The study shows evidence for divergence of a new lineage within E. canis. The location of this insertion at the 'ankyrin repeat domains' containing region is suggestive of its possible role in modulation of host responses.

Allele-specific and multiplex PCR based tools for cost-effective and comprehensive genetic testing in Congenital Adrenal Hyperplasia.

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder due to enzyme defects in adrenal steroidogenesis. Several genes code for these enzymes, out of which mutations in the CYP21A2 gene resulting in 21 hydroxylase deficiency, contribute to the most common form of CAH. However, pseudogene imposed challenges complicate genotyping CYP21A2 gene, and there is also a lack of comprehensive molecular investigations in other genetic forms of CAH in India. Here, we describe a cost-effective, highly specific, and sensitive Allele Specific PCR (ASPCR) assay designed and optimized in-house to screen eight common pathogenic mutations in the CYP21A2 gene. We have also established and utilized a multiplex PCR assay for target enrichment and Next-generation sequencing (NGS) of CYP11B1, CYP17A1, POR, and CYP19A1 genes. Following preliminary amplification of the functional gene CYP21A2, ASPCR based genotyping of eight common mutations - P30L, I2G, 8BPdel, I172N, E6CLUS (I235N, V236E, M238K) V281L, Q318X, and R356W was carried out. These results were further validated using Sanger and Next-generation sequencing. Once optimized to be specific and sensitive, the advantage of ASPCR in CYP21A2 genotyping extends to provide genetic screening for both adult and paediatric subjects and carrier testing at a low cost and less time. Furthermore, multiplex PCR coupled NGS has shown to be cost-effective and robust for parallel multigene sequencing in CAH.

A mathematical model to estimate percentage secondary infections from margin of error of diagnostic sensitivity: Useful tool for regulatory agencies to assess the risk of propagation due to false negative outcome of diagnostics

False negative outcome of a diagnosis is one the major reasons for the dissemination of the diseases with high risk of propagation. Diagnostic sensitivity and the margin of error determine the false negative outcome of the diagnosis. A mathematical model had been developed to estimate the mean % secondary infections based on the margin of error of diagnostic sensitivity, % prevalence and R0 value. This model recommends a diagnostic test with diagnostic sensitivity [≥] 96% and at least 92% lower bound limit of the 95% CI or [≤] 4% margin of error for a highly infectious diseases like COVID-19 to curb the secondary transmission of the infection due to false negative cases. Positive relationship was found between mean % secondary infection and margin of error of sensitivity suggesting greater the margin of error of a diagnostic test sensitivity, higher the number of secondary infections in a population due to false negative cases. Negative correlation was found between number of COVID-19 test kits (>90% sensitivity) with regulatory approval and margin of error (R= -0.92, p=0.023) suggesting lesser the margin of error of a diagnostic test, higher the chances of getting approved by the regulatory agencies. However, there are no specific regulatory standards available for margin of error of the diagnostic sensitivity of COVID-19 diagnostic tests. Highly infectious disease such as COVID-19, certainly need specific regulatory standards on margin of error or 95% CI of the diagnostic sensitivity to curb the dissemination of the disease due to false negative cases and our model can be used to set the standards such as sensitivity, margin of error or lower bound limit of 95% CI.