The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface ...proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7-72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in ...individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.
Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin ...combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the plasmepsin 2 and plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance.
To accurately and quickly determine the presence of copy number variations in the plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of plasmepsin 2 in Pursat in 2013 to three copies in 2014-2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015.
The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 amplifications. This study shows increasing levels of plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
Salmonellosis causes significant morbidity and mortality in Africa. Information on lineages of invasive
Salmonella
circulating in Nigeria is sparse.
Methods
Salmonella enterica
isolated ...from blood (n = 60) and cerebrospinal fluid (CSF, n = 3) between 2016 and 2020 from five tertiary hospitals in southwest Nigeria were antimicrobial susceptibility-tested and Illumina-sequenced. Genomes were analysed using publicly-available bioinformatic tools.
Results
Isolates and sequence types (STs) from blood were
S
. Typhi ST1, n = 1 and ST2, n = 43 and invasive non-typhoidal
Salmonella
(iNTS) (
S
.
Enteritidis
ST11, n = 7,
S
. Durham ST10, n = 2,
S
. Rissen ST8756, n = 2,
S
. Chester ST2063, n = 1,
S
. Dublin ST10, n = 1,
S
. Infantis ST603, n = 1,
S
. Telelkebir ST8757, n = 1 and
S
. Typhimurium ST313, n = 1).
S
. Typhi ST2 (n = 2) and
S
. Adabraka ST8757 (n = 1) were recovered from CSF. Most
S
. Typhi belonged to genotype 3.1.1 (n = 44), carried an IncY plasmid, had several antibiotic resistance genes (ARGs) including
bla
TEM-1
(n = 38),
aph(6)-Id
(n = 32),
tet(A)
(n = 33),
sul2
(n = 32),
dfrA14
(n = 30) as well as quinolone resistance-conferring gyrA_S83Y single-nucleotide polymorphisms (n = 37). All
S
. Enteritidis harboured
aph(3”)-Ib
,
bla
TEM-1
,
catA1
,
dfrA7
,
sul1
,
sul2
,
tet(B)
genes, and a single ARG,
qnrB19
, was detected in
S
. Telelkebir. Typhoidal toxins
cdtB
,
pltA
and
pltB
were detected in
S
. Typhi, Rissen, Chester, and Telelkebir.
Conclusion
Most invasive salmonelloses in southwest Nigeria are vaccine-preventable infections due to multidrug-resistant, West African dominant
S
. Typhi lineage 3.1.1. Invasive NTS serovars, including some harbouring typhoidal toxin or resistance genes, represented a third of the isolates emphasizing the need for better diagnosis and surveillance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The global spread of antimicrobial resistance (AMR) and lack of novel alternative treatments have been declared a global public health emergency by WHO. The greatest impact of AMR is experienced in ...resource-poor settings, because of lack of access to alternative antibiotics and because the prevalence of multidrug-resistant bacterial strains may be higher in low-income and middle-income countries (LMICs). Intelligent surveillance of AMR infections is key to informed policy decisions and public health interventions to counter AMR. Molecular surveillance using whole-genome sequencing (WGS) can be a valuable addition to phenotypic surveillance of AMR. WGS provides insights into the genetic basis of resistance mechanisms, as well as pathogen evolution and population dynamics at different spatial and temporal scales. Due to its high cost and complexity, WGS is currently mainly carried out in high-income countries. However, given its potential to inform national and international action plans against AMR, establishing WGS as a surveillance tool in LMICs will be important in order to produce a truly global picture. Here, we describe a roadmap for incorporating WGS into existing AMR surveillance frameworks, including WHO Global Antimicrobial Resistance Surveillance System, informed by our ongoing, practical experiences developing WGS surveillance systems in national reference laboratories in Colombia, India, Nigeria and the Philippines. Challenges and barriers to WGS in LMICs will be discussed together with a roadmap to possible solutions.
is a major pathogen in India causing community and nosocomial infections, but little is known about its molecular epidemiology and mechanisms of resistance in hospital settings. Here, we use ...whole-genome sequencing (WGS) to characterize 478
.
clinical isolates (393 methicillin-resistant
(MRSA) and 85 methicilin-sensitive
(MSSA) collected from 17 sentinel sites across India between 2014 and 2019. Sequencing results confirmed that sequence type 22 (ST22) (142 isolates, 29.7%), ST239 (74 isolates, 15.48%), and ST772 (67 isolates, 14%) were the most common clones. An in-depth analysis of 175 clonal complex (CC) 22 Indian isolates identified two novel ST22 MRSA lineages, both Panton-Valentine leukocidin+, both resistant to fluoroquinolones and aminoglycosides, and one harboring the the gene for toxic shock syndrome toxin 1
. A temporal analysis of 1797 CC22 global isolates from 14 different studies showed that the two Indian ST22 lineages shared a common ancestor in 1984 (95% highest posterior density HPD: 1982-1986), as well as evidence of transmission to other parts of the world. Moreover, the study also gives a comprehensive view of ST2371, a sublineage of CC22, as a new emerging lineage in India and describes it in relationship with the other Indian ST22 isolates. In addition, the retrospective identification of a putative outbreak of multidrug-resistant (MDR) ST239 from a single hospital in Bangalore that persisted over a period of 3 years highlights the need for the implementation of routine surveillance and simple infection prevention and control measures to reduce these outbreaks. To our knowledge, this is the first WGS study that characterized CC22 in India and showed that the Indian clones are distinct from the EMRSA-15 clone. Thus, with the improved resolution afforded by WGS, this study substantially contributed to our understanding of the global population of MRSA. IMPORTANCE The study conducted in India between 2014 and 2019 presents novel insights into the prevalence of MRSA in the region. Previous studies have characterized two dominant clones of MRSA in India, ST772 and ST239, using whole-genome sequencing. However, this study is the first to describe the third dominant clone, ST22, using the same approach. The ST22 Indian isolates were analyzed in-depth, leading to the discovery of two new sublineages of hospital-acquired
in India, both carrying antimicrobial resistance genes and mutations, which limit treatment options for patients. One of the newly characterized sublineages, second Indian cluster, carries the tsst-1 virulence gene, increasing the risk of severe infections. The geographic spread of the two novel lineages, both within India and internationally, could pose a global public health threat. The study also sheds light on ST2371 in India, a single-locus variant of ST22. The identification of a putative outbreak of MDR ST239 in a single hospital in Bangalore emphasizes the need for routine surveillance and simple infection prevention and control measures to reduce these outbreaks. Overall, this study significantly contributes to our understanding of the global population of MRSA, thanks to the improved resolution afforded by WGS.
: Although thousands of clinical isolates of
are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain ...polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions.
: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15
isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal
genes clusters.
: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates. We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa.
: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.
Pathogen genome sequencing directly from clinical samples is quickly gaining importance in genetic and medical research studies. However, low DNA yield from blood-borne pathogens is often a limiting ...factor. The problem worsens in extremely base-biased genomes such as the AT-rich Plasmodium falciparum. We present a strategy for whole-genome amplification (WGA) of low-yield samples from P. falciparum prior to short-read sequencing. We have developed WGA conditions that incorporate tetramethylammonium chloride for improved amplification and coverage of AT-rich regions of the genome. We show that this method reduces amplification bias and chimera formation. Our data show that this method is suitable for as low as 10 pg input DNA, and offers the possibility of sequencing the parasite genome from small blood samples.
Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence ...of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Alíele frequencies ranged between 1% and 3%. Three mutations were observed in > 1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.
For reasons that remain unknown, the Plasmodium falciparum genome has an exceptionally high AT content compared to other Plasmodium species and eukaryotes in general - nearly 80% in coding regions ...and approaching 90% in non-coding regions. Here, we examine how this phenomenon relates to genome-wide patterns of de novo mutation. Mutation accumulation experiments were performed by sequential cloning of six P. falciparum isolates growing in human erythrocytes in vitro for 4 years, with 279 clones sampled for whole genome sequencing at different time points. Genome sequence analysis of these samples revealed a significant excess of G:C to A:T transitions compared to other types of nucleotide substitution, which would naturally cause AT content to equilibrate close to the level seen across the P. falciparum reference genome (80.6% AT). These data also uncover an extremely high rate of small indel mutation relative to other species, primarily associated with repetitive AT-rich sequences, in addition to larger-scale structural rearrangements focused in antigen-coding var genes. In conclusion, high AT content in P. falciparum is driven by a systematic mutational bias and ultimately leads to an unusual level of microstructural plasticity, raising the question of whether this contributes to adaptive evolution.
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