Last data update: Sep 23, 2024. (Total: 47723 publications since 2009)
Records 1-11 (of 11 Records) |
Query Trace: Srinivasamoorthy G [original query] |
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Targeted metagenomics for clinical detection and discovery of bacterial tickborne pathogens
Kingry L , Sheldon S , Oatman S , Pritt B , Anacker M , Bjork J , Neitzel D , Strain A , Berry J , Sloan L , Respicio-Kingry L , Dietrich E , Bloch K , Moncayo A , Srinivasamoorthy G , Hu B , Hinckley A , Mead P , Kugeler K , Petersen J . J Clin Microbiol 2020 58 (11) Tickborne diseases, due to a diversity of bacterial pathogens, represent a significant and increasing public health threat throughout the northern hemisphere. A high-throughput 16S V1-V2 rDNA-based metagenomics assay was developed and evaluated using >13,000 residual samples from patients suspected of tickborne illness and >1000 controls. Taxonomic predictions for tickborne bacteria were exceptionally accurate, as independently validated by secondary testing. Overall, 881 specimens were positive for bacterial tickborne agents. Twelve tickborne bacterial species were detected, including two novel pathogens, representing a 100% increase in the number of tickborne bacteria identified compared to what was possible by initial PCR testing. In three blood specimens, two tickborne bacteria were simultaneously detected. Seven bacteria, not known to be tick-transmitted, were also confirmed unique to samples from persons suspected of tickborne illness. These results indicate 16S V1-V2 metagenomics can greatly simplify diagnosis and accelerate discovery of bacterial tickborne pathogens. |
Transcriptome analysis in rhesus macaques infected with hepatitis E virus genotype 1/3 infections and genotype 1 re-infection.
Choi YH , Zhang X , Srinivasamoorthy G , Purdy MA . PLoS One 2020 15 (9) e0237618 Hepatitis E virus (HEV) genotype 1 (gt1) and gt3 infections have distinct epidemiologic characteristics and genotype-specific molecular mechanisms of pathogenesis are not well characterized. Previously, we showed differences in immune response-related gene expression profiles of HEV gt1 and gt3 infections using qPCR. We hypothesize that HEV gt1 and gt3 infections induce transcriptome modifications contributing to disease pathogenesis. RNAseq analysis was performed using liver biopsy samples of naïve (baseline), HEV gt1, or gt3-infected rhesus macaques, and nine anti-HEV positive rhesus macaques re-inoculated with HEV gt1. All 10 primary HEV gt1/gt3 infected animals exhibited the typical course of acute viral hepatitis and cleared the infection between 27 to 67 days after inoculation. Viremic stages of HEV infection were defined as early, peak, and decline based on HEV RNA titers in daily stool specimens. During early, peak, and decline phases of infection, HEV gt1 induced 415, 417, and 1769 differentially expressed genes, respectively, and 310, 678, and 388 genes were differentially expressed by HEV gt3, respectively (fold change ≥ 2.0, p-value ≤ 0.05). In the HEV gt1 infection, genes related to metabolic pathways were differentially expressed during the three phases of infection. In contrast, oxidative reduction (early phase), immune responses (peak phase), and T cell cytokine production (decline phase) were found to be regulated during HEV gt3 infection. In addition, FoxO and MAPK signaling pathways were differentially regulated in re-infected and protected animals against HEV gt1 reinfection, respectively. Significant differences of hepatic gene regulation exist between HEV gt1 and gt3 infections. These findings reveal a new link between molecular pathogenesis and epidemiological characteristics seen in HEV gt1 and gt3 infections. |
Entropy of mitochondrial DNA circulating in blood is associated with hepatocellular carcinoma.
Campo DS , Nayak V , Srinivasamoorthy G , Khudyakov Y . BMC Med Genomics 2019 12 74 BACKGROUND: Ultra-Deep Sequencing (UDS) enabled identification of specific changes in human genome occurring in malignant tumors, with current approaches calling for the detection of specific mutations associated with certain cancers. However, such associations are frequently idiosyncratic and cannot be generalized for diagnostics. Mitochondrial DNA (mtDNA) has been shown to be functionally associated with several cancer types. Here, we study the association of intra-host mtDNA diversity with Hepatocellular Carcinoma (HCC). RESULTS: UDS mtDNA exome data from blood of patients with HCC (n = 293) and non-cancer controls (NC, n = 391) were used to: (i) measure the genetic heterogeneity of nucleotide sites from the entire population of intra-host mtDNA variants rather than to detect specific mutations, and (ii) apply machine learning algorithms to develop a classifier for HCC detection. Average total entropy of HCC mtDNA is 1.24-times lower than of NC mtDNA (p = 2.84E-47). Among all polymorphic sites, 2.09% had a significantly different mean entropy between HCC and NC, with 0.32% of the HCC mtDNA sites having greater (p < 0.05) and 1.77% of the sites having lower mean entropy (p < 0.05) as compared to NC. The entropy profile of each sample was used to further explore the association between mtDNA heterogeneity and HCC by means of a Random Forest (RF) classifier The RF-classifier separated 232 HCC and 232 NC patients with accuracy of up to 99.78% and average accuracy of 92.23% in the 10-fold cross-validation. The classifier accurately separated 93.08% of HCC (n = 61) and NC (n = 159) patients in a validation dataset that was not used for the RF parameter optimization. CONCLUSIONS: Polymorphic sites contributing most to the mtDNA association with HCC are scattered along the mitochondrial genome, affecting all mitochondrial genes. The findings suggest that application of heterogeneity profiles of intra-host mtDNA variants from blood may help overcome barriers associated with the complex association of specific mutations with cancer, enabling the development of accurate, rapid, inexpensive and minimally invasive diagnostic detection of cancer. |
Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing.
Qvarnstrom Y , Wei-Pridgeon Y , Van Roey E , Park S , Srinivasamoorthy G , Nascimento FS , Moss DM , Talundzic E , Arrowood MJ . Gut Pathog 2018 10 45 Background: Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms. Results: Oocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81-99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%. Conclusions: Density gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations. |
Epidemiology of a Novel Recombinant MERS-CoV in Humans in Saudi Arabia.
Assiri AM , Midgley CM , Abedi GR , Bin Saeed A , Almasri MM , Lu X , Al-Abdely HM , Abdalla O , Mohammed M , Algarni HS , Alhakeem RF , Sakthivel SK , Nooh R , Alshayab Z , Alessa M , Srinivasamoorthy G , AlQahtani SY , Kheyami A , HajOmar WH , Banaser TM , Esmaeel A , Hall AJ , Curns AT , Tamin A , Alsharef AA , Erdman D , Watson JT , Gerber SI . J Infect Dis 2016 214 (5) 712-21 BACKGROUND: The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans. Fundamental questions about circulating viruses and transmission routes remain. METHODS: We assessed routinely collected epidemiologic data for MERS-CoV cases reported in Saudi Arabia during January 01 - June 30, 2015, and conducted a more detailed investigation of cases reported during February 2015. Available respiratory specimens were obtained for sequencing. RESULTS: During the study period, 216 MERS-CoV cases were reported. Spike gene or full genome sequences (n=17) were obtained from 99 individuals. Most (72 of 99, 73%) sequences formed a discrete, novel recombinant clade (NRC-2015), which was detected in 6 regions and became predominant by June, 2015. No clinical differences were noted between clades. Among 87 cases reported during February 2015, 13 had no recognized risks for secondary acquisition; 12 of these 13 also denied camel contact. Most viruses (8 of 9) from these 13 individuals belonged to NRC-2015. DISCUSSION: Our findings document the spread and eventual predominance of NRC-2015 in humans in Saudi Arabia during the first half of 2015. Our identification of cases without recognized risk factors but with similar virus sequences suggests that additional study is needed to better understand risk factors for MERS-CoV infection. |
Finished Annotated Genome Sequence of Burkholderia pseudomallei Strain Bp1651, a Multidrug-Resistant Clinical Isolate.
Bugrysheva JV , Sue D , Hakovirta J , Loparev VN , Knipe K , Sammons SA , Ranganathan-Ganakammal S , Changayil S , Srinivasamoorthy G , Weil MR , Tatusov RL , Gee JE , Elrod MG , Hoffmaster AR , Weigel LM . Genome Announc 2015 3 (6) Burkholderia pseudomallei strain Bp1651, a human isolate, is resistant to all clinically relevant antibiotics. We report here on the finished genome sequence assembly and annotation of the two chromosomes of this strain. This genome sequence may assist in understanding the mechanisms of antimicrobial resistance for this pathogenic species. |
Draft Genome Sequences from Cyclospora cayetanensis Oocysts Purified from a Human Stool Sample.
Qvarnstrom Y , Wei-Pridgeon Y , Li W , Nascimento FS , Bishop HS , Herwaldt BL , Moss DM , Nayak V , Srinivasamoorthy G , Sheth M , Arrowood MJ . Genome Announc 2015 3 (6) The parasite Cyclospora cayetanensis causes foodborne diarrheal illness. Here, we report draft genome sequences obtained from C. cayetanensis oocysts purified from a human stool sample. The genome assembly consists of 865 contigs with a total length of 44,563,857 bases. These sequences can facilitate the development of subtyping tools to aid outbreak investigations. |
Chlamydia psittaci Comparative Genomics Reveals Intraspecies Variations in the Putative Outer Membrane and Type III Secretion System Genes.
Wolff BJ , Morrison SS , Pesti D , Ganakammal SR , Srinivasamoorthy G , Changayil S , Weil MR , MacCannell D , Rowe L , Frace M , Ritchie BW , Dean D , Winchell J . Microbiology (Reading) 2015 161 (7) 1378-91 Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer membrane proteins (pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the pmps and T3SS genes that may partially explain differences noted in C. psittaci host infection and disease. |
Real-time loop-mediated isothermal amplification (RealAmp) for the species-specific identification of Plasmodium vivax.
Patel JC , Oberstaller J , Xayavong M , Narayanan J , Debarry JD , Srinivasamoorthy G , Villegas L , Escalante AA , Dasilva A , Peterson DS , Barnwell JW , Kissinger JC , Udhayakumar V , Lucchi NW . PLoS One 2013 8 (1) e54986 Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64 degrees C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax. |
A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi.
Lucchi NW , Poorak M , Oberstaller J , Debarry J , Srinivasamoorthy G , Goldman I , Xayavong M , da Silva AJ , Peterson DS , Barnwell JW , Kissinger J , Udhayakumar V . PLoS One 2012 7 (2) e31848 BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi. |
Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA.
Demas A , Oberstaller J , Debarry J , Lucchi NW , Srinivasamoorthy G , Sumari D , Kabanywanyi AM , Villegas L , Escalante AA , Kachur SP , Barnwell JW , Peterson DS , Udhayakumar V , Kissinger JC . J Clin Microbiol 2011 49 (7) 2411-8 Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species and detecting sub-clinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S ribosomal RNA (18S rRNA) gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species-specific, highly-sensitive and amenable to both single-step and multiplex PCR. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 & Pfr364) exist in 14-41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR reaction. Parasites are routinely detected at levels of 1-10 parasites/mul. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of approximately 100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. |
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