Accurate assessment and monitoring of the Plasmodium falciparum Kelch 13 (pfk13) gene associated with artemisinin resistance is critical to understand the emergence and spread of drug-resistant ...parasites in malaria-endemic regions. In this study, we evaluated the genomic profile of the pfk13 gene associated with artemisinin resistance in P. falciparum in Nigerian children by targeted sequencing of the pfk13 gene. Genomic DNA was extracted from 332 dried blood (DBS) spot filter paper samples from three Nigerian States. The pfk13 gene was amplified by nested polymerase chain reaction (PCR), and amplicons were sequenced to detect known and novel polymorphisms across the gene. Consensus sequences of samples were mapped to the reference gene sequence obtained from the National Center for Biotechnology Information (NCBI). Out of the 13 single nucleotide polymorphisms (SNPs) detected in the pfk13 gene, five (F451L, N664I, V487E, V692G and Q661H) have not been reported in other endemic countries to the best of our knowledge. Three of these SNPs (V692G, N664I and Q661H) and a non-novel SNP, C469C, were consistent with late parasitological failure (LPF) in two States (Enugu and Plateau States). There was no validated mutation associated with artemisinin resistance in this study. However, a correlation of our study with in vivo and in vitro phenotypes is needed to establish the functional role of detected mutations as markers of artemisinin resistance in Nigeria. This baseline information will be essential in tracking and monitoring P. falciparum resistance to artemisinin in Nigeria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rabbit Haemorrhagic Disease (RHD) causes high morbidity and mortality in rabbits and hares. Here, we report the first genomic characterization of lagovirus GI.2 virus in domestic rabbits from ...sub-Saharan Africa. We used an unbiased microbial metagenomic Next Generation Sequencing (mNGS) approach to diagnose the pathogen causing the suspected outbreak of RHD in Ibadan, Nigeria. The liver, spleen, and lung samples of five rabbits from an outbreak in 2 farms were analyzed. The mNGS revealed one full and two partial RHDV2 genomes on both farms. Phylogenetic analysis showed close clustering with RHDV2 lineages from Europe (98.6% similarity with RHDV2 in the Netherlands, and 99.1 to 100% identity with RHDV2 in Germany), suggesting potential importation. Subsequently, all the samples were confirmed by RHDV virus-specific RT-PCR targeting the VP60 gene with the expected band size of 398 bp for the five rabbits sampled. Our findings highlight the need for increased genomic surveillance of RHDV2 to track its origin, understand its diversity and to inform public health policy in Nigeria, and Sub-Saharan Africa.
In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change ...discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I
R
N
, dhps double mutant G
G
and quadruple dhfr I
R
N
+ dhps G
mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I
R
N
+ dhps G
E
and sextuple dhfr I
R
N
+ dhps G
E
G
mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.
Abstract
Background
Malaria remains a public health burden especially in Nigeria. To develop new malaria control and elimination strategies or refine existing ones, understanding parasite population ...diversity and transmission patterns is crucial.
Methods
In this study, characterization of the parasite diversity and structure of
Plasmodium falciparum
isolates from 633 dried blood spot samples in Nigeria was carried out using 12 microsatellite loci of
P. falciparum
. These microsatellite loci were amplified via semi-nested polymerase chain reaction (PCR) and fragments were analysed using population genetic tools.
Results
Estimates of parasite genetic diversity, such as mean number of different alleles (13.52), effective alleles (7.13), allelic richness (11.15) and expected heterozygosity (0.804), were high. Overall linkage disequilibrium was weak (0.006, P < 0.001). Parasite population structure was low (Fst: 0.008–0.105, AMOVA: 0.039).
Conclusion
The high level of parasite genetic diversity and low population structuring in this study suggests that parasite populations circulating in Nigeria are homogenous. However, higher resolution methods, such as the 24 SNP barcode and whole genome sequencing, may capture more specific parasite genetic signatures circulating in the country. The results obtained can be used as a baseline for parasite genetic diversity and structure, aiding in the formulation of appropriate therapeutic and control strategies in Nigeria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Typhoid fever remains a significant public health concern due to cases of mis-/overdiagnosis. Asymptomatic carriers play a role in the transmission and persistence of typhoid fever, especially among ...children, where limited data exist in Nigeria and other endemic countries. We aim to elucidate the burden of typhoid fever among healthy school-aged children using the best surveillance tool(s). In a semi-urban/urban state (Osun), 120 healthy school-aged children under 15 years were enrolled. Whole blood and fecal samples were obtained from consenting children. ELISA targeting the antigen lipopolysaccharide (LPS) and anti-LPS antibodies of
, culture, polymerase chain reaction (PCR), and next-generation sequencing (NGS) were used to analyze the samples. At least one of the immunological markers was detected in 65.8% of children, with 40.8%, 37.5%, and 39% of children testing positive for IgM, IgG, and antigen, respectively. Culture, PCR, and NGS assays did not detect the presence of
in the isolates. This study demonstrates a high seroprevalence of
in these healthy children but no carriage, indicating the inability to sustain transmission. We also demonstrate that using a single technique is insufficient for typhoid fever surveillance in healthy children living in endemic areas.
This study was designed to provide information on the genetic diversity of HIV-1 and drug resistance mutations in Nigeria, as there is limited understanding of variants circulating in the country.
We ...used an advanced next-generation sequencing platform, Primer ID, to: investigate the presence of high and low abundance drug resistance mutations; characterize preexisting Integrase Strand Transfer Inhibitor (INSTI) mutations in antiretroviral therapy (ART)-experienced but dolutegravir-naive individuals; detect recent HIV-1 infections and characterize subtype diversity from a cohort of people with HIV-1 (PWH).
HIV-1 subtype analysis revealed the predominance of CRF02_AG and subtype G in our study population. At detection sensitivity of 30% abundance, drug resistance mutations (DRMs) were identified in 3% of samples. At a sensitivity level of 10%, DRMs were identified in 27.3% of samples. We did not detect any major INSTI mutation associated with dolutegravir-resistance. Only one recent infection was detected in our study population.
Our study suggests that dolutegravir-containing antiretroviral regimens will be effective in Nigeria. Our study also further emphasizes the high genetic diversity of HIV-1 in Nigeria and that CRF02_AG and subtype G are the dominant circulating forms of HIV-1 in Nigeria. These two circulating forms of the virus are largely driving the epidemic in the country.
Lassa Virus Genetics Klitting, Raphaëlle; Mehta, Samar B; Oguzie, Judith U ...
Current topics in microbiology and immunology,
2023, Letnik:
440
Journal Article
Recenzirano
In a pattern repeated across a range of ecological niches, arenaviruses have evolved a compact four-gene genome to orchestrate a complex life cycle in a narrow range of susceptible hosts. A number of ...mammalian arenaviruses cross-infect humans, often causing a life-threatening viral hemorrhagic fever. Among this group of geographically bound zoonoses, Lassa virus has evolved a unique niche that leads to significant and sustained human morbidity and mortality. As a biosafety level 4 pathogen, direct study of the pathogenesis of Lassa virus is limited by the sparse availability, high operating costs, and technical restrictions of the high-level biocontainment laboratories required for safe experimentation. In this chapter, we introduce the relationship between genome structure and the life cycle of Lassa virus and outline reverse genetic approaches used to probe and describe functional elements of the Lassa virus genome. We then review the tools used to obtain viral genomic sequences used for phylogeny and molecular diagnostics, before shifting to a population perspective to assess the contributions of phylogenetic analysis in understanding the evolution and ecology of Lassa virus in West Africa. We finally consider the future outlook and clinical applications for genetic study of Lassa virus.
Fifty patients with unexplained fever and poor outcomes presented at Irrua Specialist Teaching Hospital (ISTH) in Edo State, Nigeria, an area endemic for Lassa fever, between September 2018 - January ...2019. After ruling out Lassa fever, plasma samples from these epidemiologically-linked cases were sent to the African Centre of Excellence for Genomics of Infectious Diseases (ACEGID), Redeemer's University, Ede, Osun State, Nigeria, where we carried out metagenomic sequencing which implicated yellow fever virus (YFV) as the etiology of this outbreak. Twenty-nine of the 50 samples were confirmed positive for YFV by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), 14 of which resulted in genome assembly. Maximum likelihood phylogenetic analysis revealed that these YFV sequences formed a tightly clustered clade more closely related to sequences from Senegal than sequences from earlier Nigerian isolates, suggesting that the YFV clade responsible for this outbreak in Edo State does not descend directly from the Nigerian YFV outbreaks of the last century, but instead reflects a broader diversity and dynamics of YFV in West Africa. Here we demonstrate the power of metagenomic sequencing for identifying ongoing outbreaks and their etiologies and informing real-time public health responses, resulting in accurate and prompt disease management and control.
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•Over 50% of parasites considered in this study harboured the mutant C72I74E75T76 haplotype of the Pfcrt gene.•As it stands, CQ cannot be re-introduced as treatment for malaria in ...Nigeria if ACT resistance were to emerge.•21.1% of parasites had the Pfmdr-1 mutant N86F184 haplotype in Nigeria which suggests an increase in prevalence.•The three ACTs considered in this study, i.e. AA, AL and DHP, are still effective antimalarials with DHP slightly superior.
The emergence and spread of Plasmodium falciparum parasites resistant to artemisinin derivatives and their partners in southeastern Asia threatens malaria control and elimination efforts, and heightens the need for an alternative therapy. We have explored the distribution of P. falciparum chloroquine resistance transporter (Pfcrt) and multidrug-resistant gene 1 (Pfmdr-1) haplotypes 10 years following adoption of artemisinin-based combination therapies in a bid to investigate the possible re-emergence of Chloroquine-sensitive parasites in Nigeria, and investigated the effect of these P. falciparum haplotypes on treatment outcomes of patients treated with artemisinin-based combination therapies. A total of 271 children aged <5 years with uncomplicated falciparum malaria were included in this study. Polymorphisms on codons 72–76 of the Pfcrt gene and codon 86 and 184 of Pfmdr-1 were determined using the high resolution melting assay. Of 240 (88.6%) samples successfully genotyped with HRM for Pfcrt, wildtype C72M74N75K76 (42.9%) and mutant C72I74E75T76 (53.8%) were observed. Also, wildtype N86Y184 (62.9%) and mutant N86F184 (21.1%), Y86Y184 (6.4%), and Y86F184 (0.4%) haplotypes of Pfmdr-1 were observed. Measures of responsiveness to ACTs were similar in children infected with P. falciparum crt haplotypes (C72I74E75T76 and C72M74N75K76) and major mdr-1 haplotypes (N86Y184, N86F184 and Y86Y184). Despite a 10 year gap since the malaria treatment policy changed to ACTs, over 50% of the P. falciparum parasites investigated in this study harboured the Chloroquine-resistant C72I74E75T76 haplotype, however this did not compromise the efficacy of artemisinin-based combination therapies. Should complete artemisinin resistance emerge from or spread to Nigeria, chloroquine might not be a good alternative therapy.
Next generation sequencing (NGS)-based studies have vastly increased our understanding of viral diversity. Viral sequence data obtained from NGS experiments are a rich source of information, these ...data can be used to study their epidemiology, evolution, transmission patterns, and can also inform drug and vaccine design. Viral genomes, however, represent a great challenge to bioinformatics due to their high mutation rate and forming quasispecies in the same infected host, bringing about the need to implement advanced bioinformatics tools to assemble consensus genomes well-representative of the viral population circulating in individual patients. Many tools have been developed to preprocess sequencing reads, carry-out
or reference-assisted assembly of viral genomes and assess the quality of the genomes obtained. Most of these tools however exist as standalone workflows and usually require huge computational resources. Here we present (
iral
enomes
asily
nalyzed), a Snakemake workflow for analyzing RNA viral genomes. VGEA enables users to map sequencing reads to the human genome to remove human contaminants, split bam files into forward and reverse reads, carry out
assembly of forward and reverse reads to generate contigs, pre-process reads for quality and contamination, map reads to a reference tailored to the sample using corrected contigs supplemented by the user's choice of reference sequences and evaluate/compare genome assemblies. We designed a project with the aim of creating a flexible, easy-to-use and all-in-one pipeline from existing/stand-alone bioinformatics tools for viral genome analysis that can be deployed on a personal computer. VGEA was built on the Snakemake workflow management system and utilizes existing tools for each step:
(Chen et al., 2018) for read trimming and read-level quality control,
(Li & Durbin, 2009) for mapping sequencing reads to the human reference genome,
(Li et al., 2009) for extracting unmapped reads and also for splitting bam files into fastq files,
(Hunt et al., 2015) for
assembly to generate contigs,
(Wymant et al., 2018) to pre-process reads for quality and contamination, then map to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences,
(Shen et al., 2016) for cleaning shiver assembly for QUAST,
(Gurevich et al., 2013) to evaluate/assess the quality of genome assemblies and
(Ewels et al., 2016) for aggregation of the results from fastp, BWA and QUAST. Our pipeline was successfully tested and validated with SARS-CoV-2 (
= 20), HIV-1 (
= 20) and Lassa Virus (
= 20) datasets all of which have been made publicly available. VGEA is freely available on GitHub at: https://github.com/pauloluniyi/VGEA under the GNU General Public License.
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