Whole-Genome Sequencing of Kaposi's Sarcoma-Associated Herpesvirus from Zambian Kaposi's Sarcoma Biopsy Specimens Reveals Unique Viral Diversity

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countries—where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed 16 unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the 16 newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization.IMPORTANCEOur results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis.

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Genomic investigation of a suspected multi-drug resistant Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa

10.1101/2020.08.06.236117 ◽

2020 ◽

Author(s):

Jennifer Cornick ◽

Patrick Musicha ◽

Chikondi Peno ◽

Ezgi Saeger ◽

Pui-ying Iroh Toh ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Sub Saharan Africa ◽

Whole Genome ◽

Drug Resistant ◽

Sub Saharan ◽

Neonatal Care Unit ◽

Suspected Outbreak

ABSTRACTA suspected outbreak of multi-drug resistant (MDR) Klebsiella pneumoniae in a Malawian neonatal unit was investigated using whole-genome sequencing. Strain-types, virulence and resistance genes of K. pneumoniae isolated from patients from the hospital over a four-year period were identified. A MDR ST340 clone was implicated as the likely outbreak cause.

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Serotyping of sub-Saharan Africa Salmonella Strains Isolated from Different Sources Using Multiplex PCR and Capillary Electrophoresis Analysis and whole Genome Sequencing

10.21203/rs.3.rs-44799/v2 ◽

2020 ◽

Author(s):

Assèta Kagambèga ◽

Lari M. Hiott ◽

David S. Boyle ◽

Elizabeth A. McMillan ◽

Kaisa Haukka ◽

...

Keyword(s):

Capillary Electrophoresis ◽

Whole Genome Sequencing ◽

Multiplex Pcr ◽

Genome Sequencing ◽

Sub Saharan Africa ◽

Whole Genome ◽

Sub Saharan ◽

Different Sources

Abstract The authors have withdrawn this preprint due to author disagreement.

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New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam

mBio ◽

10.1128/mbio.01056-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 16

Author(s):

Alison E. Mather ◽

Tu Le Thi Phuong ◽

Yunfeng Gao ◽

Simon Clare ◽

Subhankar Mukhopadhyay ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Invasive Disease ◽

Multidrug Resistant ◽

Sub Saharan Africa ◽

Whole Genome ◽

Phase 2 ◽

Serovar Typhimurium ◽

Sub Saharan

ABSTRACT Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S. Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S. Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S. Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S. Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S. Typhimurium (serovar I:4,[5],12:i:− sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S. Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations. IMPORTANCE Salmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:− ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S. Typhimurium and I:4,[5],12:i:− in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections.

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Serotyping of sub-Saharan Africa Salmonella strains isolated from poultry feces using multiplex PCR and whole genome sequencing

BMC Microbiology ◽

10.1186/s12866-021-02085-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Assèta Kagambèga ◽

Lari M. Hiott ◽

David S. Boyle ◽

Elizabeth A. McMillan ◽

Poonam Sharma ◽

...

Keyword(s):

Burkina Faso ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

The United States ◽

Sub Saharan Africa ◽

Whole Genome ◽

Chain Reaction ◽

Food Borne ◽

Polymerase Chain ◽

Sub Saharan

Abstract Background Salmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 111 strains of Salmonella isolated from poultry feces in Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0. Results Among the 111 Salmonella isolates, serotypes for 17 (15.31%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. Forty-four (44) new SMART codes were developed for common and uncommon serotypes. A total of 105 (94.59%) isolates were serotyped using SeqSero 2.0 for serovar prediction based on WGS data. Conclusion We determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

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Serotyping of sub-Saharan Africa Salmonella Strains Isolated from Different Sources Using Multiplex PCR and Capillary Electrophoresis Analysis and whole Genome Sequencing

10.21203/rs.3.rs-44799/v1 ◽

2020 ◽

Author(s):

Assèta Kagambèga ◽

Lari M. Hiott ◽

David S. Boyle ◽

Elizabeth A. McMillan ◽

Kaisa Haukka ◽

...

Keyword(s):

Burkina Faso ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

The United States ◽

Sub Saharan Africa ◽

Whole Genome ◽

Food Borne ◽

Polymerase Chain ◽

Sub Saharan ◽

Different Sources

Abstract BackgroundSalmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 225 clinical, environmental, food and veterinary isolates of Salmonella from Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0.ResultsAmong the 225 Salmonella isolates, serotypes for 48 (21.33%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. One hundred and seventy-six new SMART codes were developed for common and uncommon serotypes. Serotypes for a total of 205 (91.1%) isolates were identified using SeqSero 2.0 for serovar prediction based on WGS data.ConclusionWe determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

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The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) gH/gL Complex Is the Predominant Neutralizing Antigenic Determinant in KSHV-Infected Individuals

Viruses ◽

10.3390/v12030256 ◽

2020 ◽

Vol 12 (3) ◽

pp. 256 ◽

Cited By ~ 2

Author(s):

Yasaman Mortazavi ◽

Salum J. Lidenge ◽

Tara Tran ◽

John T. West ◽

Charles Wood ◽

...

Keyword(s):

Antigenic Determinant ◽

Neutralizing Antibodies ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Sub Saharan Africa ◽

People Living With Hiv ◽

Viral Glycoproteins ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Sub Saharan ◽

Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), one of the most prevalent cancers of people living with HIV/AIDS in sub-Saharan Africa. The seroprevalence for KSHV is high in the region, and no prophylactic vaccine against the virus is available. In this study, we characterized the antigenic targets of KSHV-specific neutralizing antibodies (nAbs) in asymptomatic KSHV-infected individuals and KS patients with high nAbs titers. We quantified the extent to which various KSHV envelope glycoproteins (gB, ORF28, ORF68, gH, gL, gM, gN and gpK8.1) adsorbed/removed KSHV-specific nAbs from the plasma of infected individuals. Our study revealed that plasma from a majority of KSHV neutralizers recognizes multiple viral glycoproteins. Moreover, the breadth of nAbs responses against these viral glycoproteins varies among endemic KS, epidemic KS and asymptomatic KSHV-infected individuals. Importantly, among the KSHV glycoproteins, the gH/gL complex, but neither gH nor gL alone, showed the highest adsorption of KSHV-specific nAbs. This activity was detected in 80% of the KSHV-infected individuals regardless of their KS status. The findings suggest that the gH/gL complex is the predominant antigenic determinant of KSHV-specific nAbs. Therefore, gH/gL is a potential target for development of KSHV prophylactic vaccines.

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217 Real-world chemotherapy adherence for Kaposi’s sarcoma in Sub-Saharan Africa

Journal of Investigative Dermatology ◽

10.1016/j.jid.2019.03.293 ◽

2019 ◽

Vol 139 (5) ◽

pp. S37 ◽

Cited By ~ 1

Author(s):

L. Oyesiku ◽

S. Regan ◽

N. Busakhala ◽

F. Asirwa ◽

M. Wenger ◽

...

Keyword(s):

Real World ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Sub Saharan Africa ◽

Sub Saharan

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Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India

Microbiology Resource Announcements ◽

10.1128/mra.01388-18 ◽

2019 ◽

Vol 8 (12) ◽

Author(s):

Sivakumar Shanmugam ◽

Narender Kumar ◽

Dina Nair ◽

Mohan Natrajan ◽

Srikanth Prasad Tripathy ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

South India ◽

Sequence Data ◽

Whole Genome ◽

Drug Resistant ◽

Resistance Mutations ◽

Content Type ◽

Extensively Drug Resistant

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.

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Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: An inter-laboratory study

10.1101/793885 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ronan M. Doyle ◽

Denise M. O’Sullivan ◽

Sean D. Aller ◽

Sebastian Bruchmann ◽

Taane Clark ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Laboratory Study ◽

Clinical Microbiology ◽

Sequence Data ◽

Clinical Samples ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

AbstractBackgroundAntimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data sequenced from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants and identify problem cases and factors that lead to discordant results.MethodsWe produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their own pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime.ResultsIndividual participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment a different antibiotic would have been recommended for each isolate by at least one participant.ConclusionsWe found that participants produced discordant predictions from identical WGS data. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases and standardisation in the comparisons between genotype and resistance phenotypes will be fundamental before AST prediction using WGS can be successfully implemented in standard clinical microbiology laboratories.

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High coverage whole genome sequencing of the expanded 1000 Genomes Project cohort including 602 trios

10.1101/2021.02.06.430068 ◽

2021 ◽

Cited By ~ 4

Author(s):

Marta Byrska-Bishop ◽

Uday S. Evani ◽

Xuefang Zhao ◽

Anna O. Basile ◽

Haley J. Abel ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sequence Data ◽

Whole Genome ◽

1000 Genomes Project ◽

Phase 3 ◽

High Coverage ◽

Entire Cohort ◽

1000 Genomes ◽

Low Coverage

ABSTRACTThe 1000 Genomes Project (1kGP), launched in 2008, is the largest fully open resource of whole genome sequencing (WGS) data consented for public distribution of raw sequence data without access or use restrictions. The final (phase 3) 2015 release of 1kGP included 2,504 unrelated samples from 26 populations, representing five continental regions of the world and was based on a combination of technologies including low coverage WGS (mean depth 7.4X), high coverage whole exome sequencing (mean depth 65.7X), and microarray genotyping. Here, we present a new, high coverage WGS resource encompassing the original 2,504 1kGP samples, as well as an additional 698 related samples that result in 602 complete trios in the 1kGP cohort. We sequenced this expanded 1kGP cohort of 3,202 samples to a targeted depth of 30X using Illumina NovaSeq 6000 instruments. We performed SNV/INDEL calling against the GRCh38 reference using GATK’s HaplotypeCaller, and generated a comprehensive set of SVs by integrating multiple analytic methods through a sophisticated machine learning model, upgrading the 1kGP dataset to current state-of-the-art standards. Using this strategy, we defined over 111 million SNVs, 14 million INDELs, and ∼170 thousand SVs across the entire cohort of 3,202 samples with estimated false discovery rate (FDR) of 0.3%, 1.0%, and 1.8%, respectively. By comparison to the low-coverage phase 3 callset, we observed substantial improvements in variant discovery and estimated FDR that were facilitated by high coverage re-sequencing and expansion of the cohort. Specifically, we called 7% more SNVs, 59% more INDELs, and 170% more SVs per genome than the phase 3 callset. Moreover, we leveraged the presence of families in the cohort to achieve superior haplotype phasing accuracy and we demonstrate improvements that the high coverage panel brings especially for INDEL imputation. We make all the data generated as part of this project publicly available and we envision this updated version of the 1kGP callset to become the new de facto public resource for the worldwide scientific community working on genomics and genetics.

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